SWI5 gene as a diagnostic target for the identification of fungal and yeast species

ABSTRACT

The invention relates to the SWI5 gene, the corresponding RNA, specific probes, primers and oligonucleotides related thereto and their use in diagnostic assays to detect and/or discriminate between fungal and yeast species.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §371 of PCTInternational Application No. PCT/EP2009/057346, filed Jun. 15, 2009,which in turn claims priority to Irish Application No. 2008/0487, filedJun. 13, 2008, the contents of each which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to nucleic acid primers and probes for usein the identification of one or more fungal and yeast species. Morespecifically the invention relates to the SWI5 gene, the correspondingRNA, specific probes, primers and oligonucleotides related thereto andtheir use in diagnostic assays to detect and/or discriminate betweenfungal and yeast species.

BACKGROUND TO THE INVENTION

Yeast and fungal infections represent a major cause of morbidity andmortality among immunocompromised patients. The number ofimmunocompromised patients at risk of yeast and fungal infectioncontinues to increase each year, as does the spectrum of fungal andyeast agents causing disease. Mortality from fungal infections,particularly invasive fungal infections, is 30% or greater in certainrisk groups. The array of available anti-fungal agents is growing;however, so too is the recognition of both intrinsic and emergingresistance to antifungal drugs. These factors are contributing to theincreased need for cost containment in laboratory testing and have ledto laboratory consolidation in testing procedures.

Invasive fungal infections are on the increase. In 2003, it wasestimated that there were 9 million at risk patients of which 1.2million developed infection. Candida spp. and Aspergillus spp. now rankas the most prominent pathogens infecting immunosupressed patients. Inparticular, infections are common in the urinary tract, the respiratorysystem and the bloodstream, at the site of insertion of stents,catheters and orthopaedic joints. Approximately, 10% of the knownCandida spp. have been implicated in human infection. Invasivecandidiasis occurs when candida enters the bloodstream and it isestimated to occur at a frequency of 8/100,000 population in the US witha mortality rate of 40%. Candida albicans is the 4^(th) most commoncause of bloodstream infection. Aspergillosis usually begins as apulmonary infection that can progress to a life-threatening invasiveinfection in some patients and has a mortality rate of greater than 90%.Emerging mycoses agents include Fusarium, Scedosporium, Zygomycetes andTrichosporon spp. (“Stakeholder Insight: Invasive fungal infections”,Datamonitor, January 2004).

Immunocompromised patients including transplant and surgical patients,neonates, cancer patients, diabetics and those with HIV/AIDs are at highrisk of developing invasive fungal infections (Datamonitor report:Stakeholder opinion—Invasive fungal infections, options outweighreplacements 2004). A large number of severe cases of sepsis arereported each year. Despite improvements in its medical management,sepsis still constitutes one of the greatest challenges in intensivecare medicine. Microorganisms (bacteria, fungi and yeast) responsiblefor causing sepsis are traditionally detected in hospital laboratorieswith the aid of microbiological culture methods with poor sensitivity(25-82%), which are very time-consuming, generally in taking from two tofive days to complete, and up to eight days for the diagnosis of fungalinfections. Definitive diagnosis of infection caused by yeasts or fungiis usually based on either, the recovery and identification of aspecific agent from clinical specimens or microscopic demonstration offungi with distinct morphological features. However, there are numerouscases where these methods fail to provide conclusive proof as to theinfecting agent. In these instances, the detection of specific hostantibody responses can be used, although again this can be affected bythe immune status of the patient. Time is critical in the detection andidentification of bloodstream infections typically caused by bacteriaand fungi. Effective treatment depends on finding the source ofinfection and making appropriate decisions about antibiotics orantifungals quickly and efficiently. Only after pathogens are correctlyidentified can targeted therapy using a specific antibiotic oranti-fungal begin. Many physicians would like to see the development ofbetter in vitro amplification and direct detection diagnostic techniquesfor the early diagnosis of yeast and fungi (“Stakeholder Insight:Invasive fungal infections”, Datamonitor, January 2004). Recently Roche™launched a real time PCR based assay (Septifast™), for the detection ofbacterial, fungal and yeast DNA in clinical samples. Therefore, there isa clear need for the development of novel rapid diagnostic tests forclinically significant bacterial and fungal pathogens for bioanalysisapplications in the clinical sector. This has led the current inventorsto identify novel fungal and yeast nucleic acid targets for applicationin Nucleic Acid Diagnostics (NAD) tests. Fungal and yeast nucleic acidbased diagnostics have focused heavily on the ribosomal RNA (rRNA)genes, RNA transcripts, and their associated DNA/RNA regions. The rRNAgenes are highly conserved in all fungal species and they also containdivergent and distinctive intergenic transcribed spacer regions.Ribosomal rRNA comprises three genes: the large sub-unit gene (28S), thesmall sub-unit gene (18S) and the 5.8S gene. The 28S and 18S rRNA genesare separated by the 5.8S rRNA and two internal transcribed spacers(ITS1 and ITS2). Because the ITS region contains a high number ofsequence polymorphisms, numerous researchers have concentrated theirefforts on these as targets (Atkins and Clark, 2004). rRNA genes arealso multicopy genes with >10 copies within the fungal genome.

A number of groups are working on developing new assays for fungal andyeast infections. US2004044193 relates to, amongst a number of otheraspects, the transcription factor CaTEC1 of Candida albicans; inhibitorsthereof, and methods for the diagnosis and therapy of diseases which areconnected with a Candida infection; and also diagnostic andpharmaceutical compositions which contain the nucleotide sequences,proteins, host cells and/or antibodies. WO0183824 relates tohybridization assay probes and accessory oligonucleotides for detectingribosomal nucleic acids from Candida albicans and/or Candidadubliniensis. US6017699 and U.S. Pat. No. 5,426,026 relate to a set ofDNA primers, which can be used to amplify and speciate DNA from fivemedically important Candida species. U.S. Pat. No. 6,747,137 disclosessequences useful for diagnosis of Candida infections. EP 0422872 andU.S. Pat. No. 5,658,726 disclose probes based on 18S rRNA genes, andU.S. Pat. No. 5,958,693 discloses probes based on 28S rRNA, fordiagnosis of a range of yeast and fungal species. U.S. Pat. No.6,017,366 describes sequences based on chitin synthase gene for use innucleic acid based diagnostics for a range of Candida species. It isclear though, that development of faster, more accurate diagnosticmethods are required, particularly in light of the selection pressurecaused by modern anti-microbial treatments which give rise to increasedpopulations of resistant virulent strains with mutated genome sequences.Methods that enable early diagnosis of microbial causes of infectionenable the selection of a specific narrow spectrum antibiotic orantifungal to treat the infection (Datamonitor report: Stakeholderopinion—Invasive fungal infections, options outweigh replacements 2004;Datamonitor report: Stakeholder Opinion-Sepsis, under reaction to anoverreaction, 2006).

SWI5 is a transcription factor that activates genes involved inmitosis/Gap 1 (interphase) switch and is expressed in G1 phase of thecell cycle (Butler and Thiele 1991; Aerne et al., 1998; Akamatsu et al.,2003; Ellermeier et al., 2004; MacCallum et al., 2006). There are 128SWI5 sequences available in NCBI GenBank database including sequencesfor 6 Aspergillus spp. SWI5 sequences and 1 SWI5 sequence forNeosartorya fischeri. PCR primers were designed and applied to generatesequence information for the SWI5 gene in Aspergillus spp. SWI5 ispresent in some Candida spp. e.g. C. glabrata but not others e.g. C.albicans (MacCallum et al., 2006). Therefore, the potential exists touse SWI5 for the molecular identification of selected Candida spp.

DEFINITIONS

“Synthetic oligonucleotide” refers to molecules of nucleic acid polymersof 2 or more nucleotide bases that are not derived directly from genomicDNA or live organisms. The term synthetic oligonucleotide is intended toencompass DNA, RNA, and DNA/RNA hybrid molecules that have beenmanufactured chemically, or synthesized enzymatically in vitro.

An “oligonucleotide” is a nucleotide polymer having two or morenucleotide subunits covalently joined together. Oligonucleotides aregenerally about 10 to about 100 nucleotides. The sugar groups of thenucleotide subunits may be ribose, deoxyribose, or modified derivativesthereof such as OMe. The nucleotide subunits may be joined by linkagessuch as phosphodiester linkages, modified linkages or by non-nucleotidemoieties that do not prevent hybridization of the oligonucleotide to itscomplementary target nucleotide sequence. Modified linkages includethose in which a standard phosphodiester linkage is replaced with adifferent linkage, such as a phosphorothioate linkage, amethylphosphonate linkage, or a neutral peptide linkage. Nitrogenousbase analogs also may be components of oligonucleotides in accordancewith the invention. A “target nucleic acid” is a nucleic acid comprisinga target nucleic acid sequence. A “target nucleic acid sequence,”“target nucleotide sequence” or “target sequence” is a specificdeoxyribonucleotide or ribonucleotide sequence that can be hybridized toa complementary oligonucleotide.

An “oligonucleotide probe” is an oligonucleotide having a nucleotidesequence sufficiently complementary to its target nucleic acid sequenceto be able to form a detectable hybrid probe:target duplex under highstringency hybridization conditions. An oligonucleotide probe is anisolated chemical species and may include additional nucleotides outsideof the targeted region as long as such nucleotides do not preventhybridization under high stringency hybridization conditions.Non-complementary sequences, such as promoter sequences, restrictionendonuclease recognition sites, or sequences that confer a desiredsecondary or tertiary structure such as a catalytic active site can beused to facilitate detection using the invented probes. Anoligonucleotide probe optionally may be labelled with a detectablemoiety such as a radioisotope, a fluorescent moiety, a chemiluminescent,a nanoparticle moiety, an enzyme or a ligand, which can be used todetect or confirm probe hybridization to its target sequence.Oligonucleotide probes are preferred to be in the size range of fromabout 10 to about 100 nucleotides in length, although it is possible forprobes to be as much as and above about 500 nucleotides in length, orbelow 10 nucleotides in length.

A “hybrid” or a “duplex” is a complex formed between two single-strandednucleic acid sequences by Watson-Crick base pairings or non-canonicalbase pairings between the complementary bases. “Hybridization” is theprocess by which two complementary strands of nucleic acid combine toform a double-stranded structure (“hybrid” or “duplex”). A “fungus” or“yeast” is meant any organism of the kingdom Fungi, and preferably, isdirected towards any organism of the phylum Ascomycota.

“Complementarity” is a property conferred by the base sequence of asingle strand of DNA or RNA which may form a hybrid or double-strandedDNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding betweenWatson-Crick base pairs on the respective strands. Adenine (A)ordinarily complements thymine (T) or uracil (U), while guanine (G)ordinarily complements cytosine (C).

The term “stringency” is used to describe the temperature, ionicstrength and solvent composition existing during hybridization and thesubsequent processing steps. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying those parameterseither individually or together. Under high stringency conditions onlyhighly complementary nucleic acid hybrids will form; hybrids without asufficient degree of complementarity will not form. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity needed between two nucleic acid strands forming ahybrid. Stringency conditions are chosen to maximize the difference instability between the hybrid formed with the target and the non-targetnucleic acid. With “high stringency” conditions, nucleic acid basepairing will occur only between nucleic acid fragments that have a highfrequency of complementary base sequences (for example, hybridizationunder “high stringency” conditions, may occur between homologs withabout 85-100% identity, preferably about 70-100% identity). With mediumstringency conditions, nucleic acid base pairing will occur betweennucleic acids with an intermediate frequency of complementary basesequences (for example, hybridization under “medium stringency”conditions may occur between homologs with about 50-70% identity). Thus,conditions of “weak” or “low” stringency are often required with nucleicacids that are derived from organisms that are genetically diverse, asthe frequency of complementary sequences is usually less.

‘High stringency’ conditions are those equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, ph adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length is used.

“Medium stringency’ conditions are those equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0%SDS at 42° C., when a probe of about 500 nucleotides in length is used.‘Low stringency’ conditions are those equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml:5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C., when a probe of about 500nucleotides in length is used.

In the context of nucleic acid in-vitro amplification basedtechnologies, “stringency” is achieved by applying temperatureconditions and ionic buffer conditions that are particular to thatin-vitro amplification technology. For example, in the context of PCRand real-time PCR, “stringency” is achieved by applying specifictemperatures and ionic buffer strength for hybridisation of theoligonucleotide primers and, with regards to real-time PCR hybridisationof the probe/s, to the target nucleic acid for in-vitro amplification ofthe target nucleic acid.

One skilled in the art will understand that substantially correspondingprobes of the invention can vary from the referred-to sequence and stillhybridize to the same target nucleic acid sequence. This variation fromthe nucleic acid may be stated in terms of a percentage of identicalbases within the sequence or the percentage of perfectly complementarybases between the probe and its target sequence. Probes of the presentinvention substantially correspond to a nucleic acid sequence if thesepercentages are from about 100% to about 80% or from 0 base mismatchesin about 10 nucleotide target sequence to about 2 bases mismatched in anabout 10 nucleotide target sequence. In preferred embodiments, thepercentage is from about 100% to about 85%. In more preferredembodiments, this percentage is from about 90% to about 100%; in otherpreferred embodiments, this percentage is from about 95% to about 100%

By “sufficiently complementary” or “substantially complementary” ismeant nucleic acids having a sufficient amount of contiguouscomplementary nucleotides to form, under high stringency hybridizationconditions, a hybrid that is stable for detection. The terms “identical”or percent “identity,” in the context of two or more nucleic acids orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same (i.e., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or higher identity over a specified region, whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site atncbi.nlm.nih.gov/BLAST/or the like). Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1987-2005, WileyInterscience)). A preferred example of algorithm that is suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids and proteins of the invention. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

By “nucleic acid hybrid” or “probe:target duplex” is meant a structurethat is a double-stranded, hydrogen-bonded structure, preferably about10 to about 100 nucleotides in length, more preferably 14 to 50nucleotides in length, although this will depend to an extent on theoverall length of the oligonucleotide probe. The structure issufficiently stable to be detected by means such as chemiluminescent orfluorescent light detection, autoradiography, electrochemical analysisor gel electrophoresis. Such hybrids include RNA:RNA, RNA:DNA, orDNA:DNA duplex molecules.

“RNA and DNA equivalents” refer to RNA and DNA molecules having the samecomplementary base pair hybridization properties. RNA and DNAequivalents have different sugar groups (i.e., ribose versusdeoxyribose), and may differ by the presence of uracil in RNA andthymine in DNA. The difference between RNA and DNA equivalents do notcontribute to differences in substantially corresponding nucleic acidsequences because the equivalents have the same degree ofcomplementarity to a particular sequence.

By “preferentially hybridize” is meant that under high stringencyhybridization conditions oligonucleotide probes can hybridize theirtarget nucleic acids to form stable probe:target hybrids (therebyindicating the presence of the target nucleic acids) without formingstable probe:non-target hybrids (that would indicate the presence ofnon-target nucleic acids from other organisms). Thus, the probehybridizes to target nucleic acid to a sufficiently greater extent thanto non-target nucleic acid to enable one skilled in the art toaccurately detect the presence of (for example Candida) and distinguishthese species from other organisms. Preferential hybridization can bemeasured using techniques known in the art and described herein.

By “theranostics” is meant the use of diagnostic testing to diagnose thedisease, choose the correct treatment regime and monitor the patientresponse to therapy. The theranostics of the invention may be based onthe use of an NAD assay of this invention on samples, swabs or specimenscollected from the patient.

OBJECT OF THE INVENTION

It is an object of the current invention to provide sequences and/ordiagnostic assays to detect and identify one or more fungal and yeastspecies. The current inventors have used the SWI5 gene sequence todesign primers and probes that are specific to Aspergillus SWI5 genes.Such primers may allow the detection of yeast and fungal species andalso allow distinction between Candida and Aspergillus species, foeexample. The current invention further provides for primers and probesthat may allow discrimination between different Candida spp. and amongdifferent Aspergillus spp.

SUMMARY OF THE INVENTION

The present invention provides for a diagnostic kit for detection andidentification of fungal rand yeast species, comprising anoligonucleotide probe capable of binding to at least a portion of theSWI5 gene or its corresponding mRNA. The oligonucleotide probe may havea sequence substantially homologous to or substantially complementary toat least a portion of the SWI5 gene or its corresponding mRNA. It willthus be capable of binding or hybridizing with a complementary DNA orRNA molecule. The SWI5 gene may be a fungal SWI5 gene. The SWI5 gene maybe yeast SWI5 gene. The nucleic acid molecule may be synthetic. The kitmay comprise more than one such probe. In particular, the kit maycomprise a plurality of such probes. In addition the kit may compriseadditional probes for other organisms, such as, for example, bacterialspecies or viruses.

The identified sequences are suitable not only for in vitro DNA/RNAamplification based detection systems but also for signal amplificationbased detection systems. Furthermore, the sequences of the inventionidentified as suitable targets provide the advantages of havingsignificant intragenic sequence heterogeneity in some regions, which isadvantageous and enables aspects of the invention to be directed towardsgroup or species-specific targets, and also having significant sequencehomogeneity in some regions, which enables aspects of the invention tobe directed towards genus-specific fungal and yeast primers and probesfor use in direct nucleic acid detection technologies, signalamplification nucleic acid detection technologies, and nucleic acid invitro amplification technologies for fungal and yeast diagnostics. TheSWI5 sequences allow for multi-test capability and automation indiagnostic assays.

One of the advantages of the sequences of the present invention is thatthe intragenic SWI5 nucleotide sequence diversity between closelyrelated fungi and yeast enables specific primers and probes for use indiagnostics assays for the detection of fungi and yeast to be designed.The SWI5 nucleotide sequences, both DNA and RNA can be used with directdetection, signal amplification detection and in vitro amplificationtechnologies in diagnostics assays. The SWI5 sequences allow formulti-test capability and automation in diagnostic assays.

The kit may further comprise a primer for amplification of at least aportion of the SWI5 gene. Suitably, the kit comprises a forward and areverse primer for a portion of the SWI5 gene.

The portion of the SWI5 gene may be equivalent to a region of SWI5equivalent to by positions 1-2319.

Equivalent positions to base pair position 1 to base pair position 2319in A. fumigatus can be found in other organisms, but not necessarily inthe same position.

The portion of the SWI5 gene may be equivalent to two regions of theSWI5 gene in Aspergillus spp. equivalent to base pair position 38 tobase pair position 472 in A. fumigatus (region 1) and from base pairposition 1423 to 1627 base pair position in A. fumigatus (region 3).

Equivalent positions to base pair position 38 to base pair position 472and from base pair position 1423 to 1627 in A. fumigatus can be found inother organisms, but not necessarily in the same position.

The kit may also comprise additional probes.

The probe may have a sequence selected from the group SEQ ID NO 17, 18,42, 45, 48, 51, 54, 61, 64, or 67 or a sequence substantially homologousto or substantially complementary to those sequences which can also actas a probe for the SWI5 gene.

The kit may comprise at least one forward in vitro amplification primerand at least one reverse in vitro amplification primer. Such primers mayinclude a forward primer which preferentially hybridizes to the samenucleotide sequence as is preferentially hybridized by SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 36, 38, 40, 43, 46, 49, 52, 55, 58, 59, 62 or 65and/or a reverse primer which preferentially hybridizes to the samenucleotide sequence as is preferentially hybridized by SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, 37, 39, 41, 44, 47, 50, 53, 56, 57, 60, 63 or 66,or a sequence being substantially homologous or complementary theretowhich can also act as a forward or reverse amplification primer. Thediagnostic kit may be based on direct nucleic acid detectiontechnologies, signal amplification nucleic acid detection technologies,and nucleic acid in vitro amplification technologies is selected fromone or more of Polymerase Chain Reaction (PCR), Ligase Chain Reaction(LCR), Nucleic Acids Sequence Based Amplification (NASBA), StrandDisplacement Amplification (SDA), Transcription Mediated Amplification(TMA), Branched DNA technology (bDNA) and Rolling Circle AmplificationTechnology (RCAT)), or other in vitro enzymatic amplificationtechnologies.

The invention also provides a nucleic acid molecule selected from thegroup consisting of SEQ ID NO.1 to SEQ ID NO. 95 and sequencessubstantially homologous thereto, or substantially complementary to aportion thereof and having a function in diagnostics based on the SWI5gene. The nucleic acid molecule may comprise an oligonucleotide having asequence substantially homologous to or substantially complementary to aportion of a nucleic acid molecule of SEQ ID NO.1 to SEQ ID NO. 95. Theinvention also provides a method of detecting a target organism in atest sample comprising the steps of:

-   -   (i) Mixing the test sample with at least one oligonucleotide        probe as defined above under appropriate conditions; and    -   (ii) hybridizing under high stringency conditions any nucleic        acid that may be present in the test sample with the        oligonucleotide to form a probe:target duplex; and    -   (iii) determining whether a probe:target duplex is present; the        presence of the duplex positively identifying the presence of        the target organism in the test sample.

The nucleic acid molecule and kits of the present invention may be usedin a diagnostic assay to detect the presence of one or more fungaland/or yeast species, to measure fungal and/or yeast titres in a patientor in a method of assessing the efficacy of a treatment regime designedto reduce yeast and/or fungal titre in a patient or to measure fungaland/or yeast contamination in an environment. The environment may be ahospital, or it may be a food sample, an environmental sample e.g.water, an industrial sample such as an in-process sample or an endproduct requiring bioburden or quality assessment.

The kits and the nucleic acid molecule of the invention may be used inthe identification and/or characterization of one or more disruptiveagents that can be used to disrupt the SWI5 gene function. Thedisruptive agent may be selected from the group consisting of antisenseRNA, PNA, and siRNA.

In some embodiments of the invention, a nucleic acid molecule comprisinga species-specific probe can be used to discriminate between species ofthe same genus.

The oligonucleotides of the invention may be provided in a compositionfor detecting the nucleic acids of fungal and yeast target organisms.Such a composition may also comprise buffers, enzymes, detergents, saltsand so on, as appropriate to the intended use of the compositions. It isalso envisioned that the compositions, kits and methods of theinvention, while described herein as comprising at least one syntheticoligonucleotide, may also comprise natural oligonucleotides withsubstantially the same sequences as the synthetic nucleotide fragmentsin place of, or alongside synthetic oligonucleotides.

The invention also provides for an in vitro amplification diagnostic kitfor a target fungal and/or yeast organism comprising at least oneforward in vitro amplification primer and at least one reverse in vitroamplification primer, the forward amplification primer being selectedfrom the group consisting of one or more of or a sequence beingsubstantially homologous or complementary thereto which can also act asa forward amplification primer, and the reverse amplification primerbeing selected from the group consisting of one or more of or a sequencebeing substantially homologous or complementary thereto which can alsoact as a reverse amplification primer.

The invention also provides for a diagnostic kit for detecting thepresence of candidate fungal and/or yeast species, comprising one ormore DNA probes comprising a sequence substantially complementary to, orsubstantially homologous to the sequence of the SWI5 gene of thecandidate fungal and/or species.

A kit useful for detecting an Aspergillus or Candida glabrata SWI5polynucleotide comprises an oligonucleotide probe selected from SEQ IDNOs: 17, 18, 42, 45, 48, 51, 54, 61, 64, or 67 or a probe whichpreferentially hybridizes to the same nucleotide sequence as ispreferentially hybridized by SEQ ID NOs: 17, 18, 42, 45, 48, 51, 54, 61,64, or 67. The kit may further comprise a forward primer selected fromSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 36, 38, 40, 43, 46, 49, 52, 55,58, 59, 62 or 65 or a probe which preferentially hybridizes to the samenucleotide sequence as is preferentially hybridized by SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, 15, 36, 38, 40, 43, 46, 49, 52, 55, 58, 59, 62 or 65and/or a reverse primer selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12,14, 16, 37, 39, 41, 44, 47, 50, 53, 56, 57, 60, 63 or 66 or a probewhich preferentially hybridizes to the same nucleotide sequence as ispreferentially hybridized by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 37,39, 41, 44, 47, 50, 53, 56, 57, 60, 63 or 66.

A kit for detecting or identifying a Aspergillus fumigatus SWI5polynucleotide comprises an oligonucleotide probe selected from SEQ IDNO: 17 or 18 or 42 and 54 or a sequence which preferentially hybridizesto the same nucleotide sequence as is preferentially hybridized by SEQID NO: 17 or 18 or 42 and 54 and further comprises a forward primerselected from SEQ ID NO: 43, 55, 58 or a sequence which preferentiallyhybridizes to the same nucleotide sequence as is preferentiallyhybridized by SEQ ID NO: 43, 55, 58 and a reverse primer which isselected from SEQ ID NO: 44, 56, 57 or a sequence which preferentiallyhybridizes to the same nucleotide sequence as is preferentiallyhybridized by SEQ ID NO: 44, 56, 57.

A kit for detecting or identifying a Aspergillus flavus SWI5polynucleotide comprises an oligonucleotide probe selected from SEQ IDNO: 45, 61 and sequences which preferentially hybridizes to the samenucleotide sequence as is preferentially hybridized by SEQ ID NO: 45, 61and further comprises a forward primer selected from SEQ ID NO: 46, 59and sequences which preferentially hybridizes to the same nucleotidesequence as is preferentially hybridized by SEQ ID NO: 46, 59 and areverse primer selected from SEQ ID NO: 47, 60 and sequences whichpreferentially hybridizes to the same nucleotide sequence as ispreferentially hybridized by SEQ ID NO: 47, 60.

A kit for detecting or identifying a Aspergillus niger SWI5polynucleotide comprises an oligonucleotide probe selected from SEQ IDNO: 48, 67 and sequences which preferentially hybridizes to the samenucleotide sequence as is preferentially hybridized by SEQ ID NO: 48, 67and further comprises a forward primer selected from SEQ ID NO: 48, 65and sequences which preferentially hybridizes to the same nucleotidesequence as is preferentially hybridized by SEQ ID NO: 48, 65 and areverse primer selected from SEQ ID NO: 49, 66 and sequences whichpreferentially hybridizes to the same nucleotide sequence as ispreferentially hybridized by SEQ ID NO: 49, 66.

A kit for detecting or identifying a Aspergillus terreus SWI5polynucleotide comprises an oligonucleotide probe selected from SEQ IDNO: 51, 64 and sequences which preferentially hybridizes to the samenucleotide sequence as is preferentially hybridized by SEQ ID NO: 51, 64and further comprises a forward primer selected from SEQ ID NO: 52, 62and sequences which preferentially hybridizes to the same nucleotidesequence as is preferentially hybridized by SEQ ID NO: 52, 62 and areverse primer selected from SEQ ID NO: 53, 63 and sequences whichpreferentially hybridizes to the same nucleotide sequence as ispreferentially hybridized by SEQ ID NO: 53, 63.

The present invention also provides for one or more syntheticoligonucleotides having a nucleotide sequence substantially homologousto or substantially complementary to one or more of the group consistingof the SWI5 gene or mRNA transcript thereof, the fungal SWI5 gene ormRNA transcript thereof, the yeast SWI5 gene or mRNA transcript thereof,one or more of SEQ ID NO 1-SEQ ID NO 95.

The nucleotide may comprise DNA. The nucleotide may comprise RNA. Thenucleotide may comprise a mixture of DNA, RNA and PNA. The nucleotidemay comprise synthetic nucleotides. The sequences of the invention (andthe sequences relating to the methods, kits compositions and assays ofthe invention) may be selected to be substantially homologous to aportion of the coding region of the SWI5 gene. The gene may be a genefrom a target fungal and/or yeast organism. The sequences of theinvention are preferably sufficient so as to be able to form aprobe:target duplex to the portion of the sequence.

The invention also provides for a diagnostic kit for a target fungal oryeast organism comprising an oligonucleotide probe substantiallyhomologous to or substantially complementary to an oligonucleotide ofthe invention (which may be synthetic). It will be appreciated thatsequences suitable for use as in vitro amplification primers may also besuitable for use as oligonucleotide probes: while it is preferable thatamplification primers may have a complementary portion of between about15 nucleotides and about 30 nucleotides (more preferably about 15-about23, most preferably about 20 to about 23), oligonucleotide probes of theinvention may be any suitable length. The skilled person will appreciatethat different hybridization and or annealing conditions will berequired depending on the length, nature & structure (eg. Hybridizationprobe pairs for LightCycler, Taqman 5′ exonuclease probes, hairpin loopstructures etc. and sequence of the oligonucleotide probe selected.

Kits and assays of the invention may also be provided wherein theoligonucleotide probe is immobilized on a surface. Such a surface may bea bead, a membrane, a column, dipstick, a nanoparticle, the interiorsurface of a reaction chamber such as the well of a diagnostic plate orinside of a reaction tube, capillary or vessel or the like. The targetfungal organism may be selected from the group consisting of A.fumigatus, N. fischeri, A. clavatus, A. niger, A. terreus, A. flavus, A.versicolor and A. nidulans. The target yeast organisms may be theCandida species C. glabrata.

Under these circumstances, the amplification primers and oligonucleotideprobes of the invention may be designed to a gene specific or genusspecific region so as to be able to identify one or more, or most, orsubstantially all of the desired organisms of the target yeast organismgrouping.

The target fungal organisms may be an Aspergillus species for given setof primers already experimentally demonstrated, and more preferably,selected from the group consisting of A. fumigatus, A. clavatus, A.niger, A. terreus, A. flavus, A. versicolor and A. nidulans.

The test sample may comprise cells of the target fungal and/or yeastorganism. The method may also comprise a step for releasing nucleic acidfrom any cells of the target fungal or yeast organism that may bepresent in said test sample. Ideally, the test sample is a lysate of anobtained sample from a patient (such as a swab, or blood, urine, saliva,a bronchial lavage, dental specimen, skin specimen, scalp specimen,transplant organ biopsy, stool, mucus, or discharge sample). The testsamples may be a food sample, a water sample, an environmental sample,an end product, end product or in-process industrial sample.

The invention also provides for the use of any one of SEQ ID NO.1 to SEQID NO. 35 in a diagnostic assay for the presence of one or more yeast orfungal species. The species may be selected from the group consisting ofC. glabrata, A. fumigatus, N. fischeri, A. clavatus, A. niger, A.terreus, A. flavus, A. versicolor and A. nidulans. The invention alsoprovides for kits for use in clinical diagnostics, theranostics, foodsafety diagnostics, industrial microbiology diagnostics, environmentalmonitoring, veterinary diagnostics, bio-terrorism diagnostics comprisingone or more of the synthetic oligonucleotides of the invention. The kitsmay also comprise one or more articles selected from the groupconsisting of appropriate sample collecting instruments, reagentcontainers, buffers, labelling moieties, solutions, detergents andsupplementary solutions. The invention also provides for use of thesequences, compositions, nucleotide fragments, assays, and kits of theinvention in theranostics, Food safety diagnostics, Industrialmicrobiology diagnostics, Environmental monitoring, Veterinarydiagnostics, Bio-terrorism diagnostics.

The nucleic acid molecules, composition, kits or methods may be used ina diagnostic nucleic acid based assay for the detection of fungal and/oryeast species.

The nucleic acid molecules, composition, kits or methods may be used ina diagnostic assay to measure fungal and/or yeast titres in a patient.The titres may be measured in vitro.

The nucleic acid molecules, composition, kits or methods may be used ina method of assessing the efficacy of a treatment regime designed toreduce fungal and/or yeast titre in a patient comprising assessing thefungal and/or yeast titre in the patient (by in vivo methods or in vitromethods) at one or more key stages of the treatment regime. Suitable keystages may include before treatment, during treatment and aftertreatment. The treatment regime may comprise an antifungal agent, suchas a pharmaceutical drug. The nucleic acid molecules, composition, kitsor methods may be used in a diagnostic assay to measure potential fungaland/or yeast contamination, for example, in a hospital. The nucleic acidmolecules, composition, kits or methods may be used in theidentification and/or characterization of one or more disruptive agentsthat can be used to disrupt the SWI5 gene function. Suitable disruptiveagents may be selected from the group consisting of antisense RNA, PNA,siRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Selected primer binding sites in the SWI5 gene of Aspergillusfumigatus SEQ ID NO 29. The regions (1, 2 and 3) of interest areunderlined (Position of Region 1: 38 to 472. Position of Region 2:1034-1241. Position of Region 3: 1423-1627).

FIG. 2: Binding site of A. fumigatus probe P1-AspSWI5-1, SEQ ID NOs 17(underlined and bolded) in the amplified fragment of SWI5 (Region 1 ofinterest). PCR primes AspSWI5-1-F (SEQ ID NO. 1)/AspSWI5-1-R (SEQ ID NO.2) are highlighted.

FIG. 3: Binding site of A. fumigatus probe P1-AspSWI5-3, SEQ ID NO. 18.(underlined and bolded) in the amplified fragment of SWI5 (Region 3 ofinterest). PCR primers AspSWI5-3-F (SEQ ID NO. 5)/AspSWI5-3-R (SEQ IDNO. 6) are highlighted.

FIG. 4: Resulting amplification plot from real-time PCR assay for A.fumigatus based on region 1 of the SWI5 gene with TaqMan probeP1-AspSWI5-1. Specificity of the assay was tested against a panel of DNAfrom 6 closely related Aspergillus species and C. albicans. The 3 A.fumigatus strains tested were detected and no cross-reaction wasobserved with DNA from the other species tested.

FIG. 5: Resulting amplification plot from real-time PCR assay for A.fumigatus based on region 3 of the SWI5 gene with TaqMan probeP1-AspSWI5-3. Specificity of the assay was tested against a panel of DNAfrom 6 closely related Aspergillus species and C. albicans. The 3 A.fumigatus strains tested were detected and no cross-reaction wasobserved with DNA from the other species tested.

FIG. 6: Exclusivity of the SWi5 assays for A. fumigatus, A. flavus, A.niger and A. terreus. Exclusivity assays were preformed withthermocycling conditions which included denaturation and annealing at95° C. for 5 seconds and 60° C. for 10 seconds for 50 cycles. The probesdetected only the species for which they were designed, with nocross-reaction observed.

FIG. 7: Limit of detection of the A. fumigatus Afum_SWI5_1 assay The LODof the A. fumigatus assay Afum_SWI5_1 was preformed with annealing at95° C. for 5 seconds and 60° C. for 10 seconds for 50 cycles.

FIG. 8: Limit of detection of the SWI5 A. terreus, Aterr_SWI5_1 assay

The results show the LOD for the A. terreus Aterr_SWI5_1 assay.

The thermocycling conditions included annealing at 95° C. for 5 secondsand 60° C. for 10 seconds for 50 cycles. An LOD of 10⁵ cell equivalentsper reaction was obtained.

FIG. 9: Limit of detection of A. fumigatus Afum_SWI5_2:

Graphs show the results obtained for the LOD assays preformed on A.fumigatus Afum_SWI5_2 assay. Results shown in graph a were obtainedfollowing denaturation and annealing annealing for 95° C. for 10 secsand 60° C. for 30 secs for 50 cycles respectively. The results shown ingraph b were obtained following denaturation and annealing at 95° C. for5 secs and 60° C. for 20 secs, for 40 cycles. The LOD obtained was 100cell equivalent.

FIG. 10: Limit of detection for the SWI5_2 assays for A. flavus, A.niger and A. terreus.

Annealing was 95° C. for 5 secs and 60° C. for 10 secs, for 45 cycles.A. niger assay shown in graph c was the only successful assay with a LODof 10 cell equivalents.

FIG. 11 Master alignment of SWI5 sequence information.

DETAILED DESCRIPTION OF THE INVENTION Example 1

Materials and Methods

Cell Culture

Aspergillus species were cultured in Sabouraud broth (4% wt/vol glucose,1% wt/vol peptone, 1.5% agar) or agar for 3-4 days at 25° C.

DNA Extraction

Aspergillus spp. were pre-treated with lyticase or zymolase enzymesprior to DNA isolation. DNA was isolated from Apergillus spp. using theMagNA Pure System (Roche Molecular Systems) in combination with theMagNA pure Yeast and Bacterial isolation kit III according to themanufacturers protocol.

DNA Sequencing of SWI5 Gene Regions in Aspergillus spp.

The publicly available sequences of the SWI5 genes for Aspergillusspecies were acquired from the NCBI GenBank database and aligned usingClustal W. Combinations of PCR primers were used to amplify sub-regionsof SWI5 in Aspergillus species equivalent by 1 to by 2319 of Aspergillusfumigatus. For example, PCR Primers AspSWI5-1-F/Asp SWI5-1-R weredesigned to amplify a region in Aspergillus spp. equivalent to byposition 38 to 472 in A. fumigatus XM_749401.1 (Region 1, FIG. 1). PCRprimers AspSWI5-2-F/AspSWI5-2-R were designed to amplify a region inAspergillus spp. equivalent to by position 1034 to 1241 in A. fumigatus(Region 2, FIG. 1). AspSWI5-3-F/AspSWI5-3-R were designed to amplify aregion in Aspergillus spp. equivalent to by position 1423 to 1627 in A.fumigatus XM_749401.1 (Region 3, FIG. 1). The SWI5 gene regions wereamplified in a range of Aspergillus spp. iCycler BioRad PCR machine orthe PTC200 Peltier thermocycler (MJ Research) using the reagentsoutlined in Table 2 and the thermocycling conditions described in Table3 or modifications thereof. The PCR reaction products were purified withRoche High Pure PCR Product Purification kit or with the ExoSAP-IT kit(USB) according to the manufacturers' instructions sent for sequencingto Sequiserve, Germany and sequenced using the forward amplificationprimer AspSWI5-1-F or AspSWI5-3-F. DNA sequence information wasgenerated as follows: Aspergillus region 1 sequence information wasgenerated for 5 Aspergillus species (A. fumigatus, A. nidulans, A.clavatus, A. niger, A. flavus) and Neosartorya fischeri. Aspergillusregion 3 sequence information was generated for 3 Aspergillus species(A. fumigatus, A. nidulans, A. niger) and Neosartorya fischeri.

TABLE 1 PCR primers designed to amplify SWI5 gene regionsin Aspergillus spp. Primer Name Primer Sequence SEQ ID NO. 1 AspSWI5-1-FATCGACAACATCGTCGGCAGA SEQ ID NO. 2 AspSWI5-1-R GCTGTTGCTGTTGCATCAGATTSEQ ID NO. 3 AspSWI5-2-F TAGCCGCCATGCCAAGC SEQ ID NO. 4 AspSWI5-2-RCCAGTCTCTTTGATAGAAGCA SEQ ID NO. 5 AspSWI5-3-F CGTGGACATGACCTG AAGCSEQ ID NO. 6 AspSWI5-3-R GTCTCTCCTCCAACTCTGG SEQ ID NO. 7 1FATGTTAGCCAATCCAC SEQ ID NO. 8 1R ATTCCAGGCACCG SEQ ID NO. 9 2FCTTGAGGGCCAAATC SEQ ID NO. 10 2R CTCGTCCTTTCAATCC SEQ ID NO. 11 3FACTATGCCTCGTCG SEQ ID NO. 12 3R AGCGAATACATTGCC SEQ ID NO. 13 4FACAAACCATATGAATGTC SEQ ID NO. 14 4R GCAGGCTCGGTT SEQ ID NO. 15 5FCCTCGAGAAGATCGT SEQ ID NO. 16 5R CTAGCAGTCCATGAAG

TABLE 2 PCR reagents used to amplify the SWI5 gene regions inAspergillus spp SAMPLE PCR Reaction Mix x 1 10 x Buffer (100 mM TrisHCl, 5 μl 15 mM MgCl₂, 500 mM KCl pH 8.3) dNTP's Mix, Roche (10 mM dNTP)1 μl Primer Forward primer (10 μM) 1 μl Primer Reverse primer (10 μM) 1μl Polymerase TaqPol, Roche 1 U/μl 1 μl H₂O Amgen/Accugene 36-39 μlGenomic DNA Template 2-5 μl TOTAL VOLUME 50 μl

TABLE 3 PCR reaction conditions applied to amplify the SWI5 gene regionsin Aspergillus spp. PCR Thermal profile   Lid preheating was ON StepTemp Time 1 94° C. 1 min X 35 2 50° C.-59° C. 1 min 3 72° C. 1 min 4 72°C. 7 min 5  8° C. Hold

TABLE 5 Real-time PCR reagents Preparation of PCR Reaction MixLightCycler ® FastStartDNA Master HybProbe, SAMPLE Roche Cat. 03 003 24800 x 1 HybProb mix 10 x conc. (Red cap) 2 μl MgCl₂ stock solution (Bluecap) 1.6 μl (Final conc. in reaction is 3 mM) Probe P1-AspSWI5 orP1-AspSWI5-3 2 μl Primer Forward AspSWI5-1-F or AspSWI5-3-F 1 μl PrimerReverse AspSWI5-1-R or AspSWI5-3-R 1 μl H₂O PCR-grade 10.4 μl Template 2μl TOTAL VOLUME 20 μl

TABLE 4 TaqMan probes(5′-FAM and 3′-BHQ1 labels) based onthe SWI5 gene regions in A. fumigatus. Probe Name Probe SequenceSEQ ID NO. 17 P1-AspSWI5-1 CCAAAGTTCCTACCCTTCCAGCAC SEQ ID NO. 18P1-AspSWI5-3 CTGACTCGGCACAGACAACGAGGA

TABLE 6 Real-time PCR thermocycling conditions PCR Thermal profile CycleStep Temp Time Activation 1 95° C. 10 min X 50 Amplification 1 95° C. 10sec 2 62-65° C. 20 sec 3 70° C. 10 sec Cooling 1 40° C. HoldResultsPrimer and Probe Design

The publicly available sequence information available for the SWI5 genein Aspergillus spp. was aligned with the newly generated sequenceinformation for the SWI5 gene in Aspergillus spp. and analysed usingbioinformatics tools. Species-specific probes were designed based on thecompiled SWI5 sequence information for Aspergillus fumigatus (regions 1and 3) (Table 4). FIGS. 2 and 3 show the relative positions of the PCRprimers and TaqMan DNA probes for the amplification and detection of A.fumigatus.

Real-Time PCR

The specificity of the TaqMan probes for the identification of A.fumigatus was demonstrated in real-time PCR assays on the LightCyclerusing the reagents and thermocycling conditions outlined in Tables 5 and6. For the A. fumigatus assay based on the SWI5 gene region 1, PCRprimers AspSWI5-1-F/AspSWI5-1-R were combined in with TaqMan probe,P1-AspSWI5-1. For the A. fumigatus assay based on the SWI5 gene region3, PCR primers AspSWI5-3-F/AspSWI5-3-R were combined with TaqMan probe,P1-AspSWI5-3.

The specificity of the assays for the detection of A. fumigatus wasconfirmed by including DNA from a range of closely related Aspergillusspecies and C. albicans in the A. fumigatus real-time PCR assays. Theassays detected A. fumigatus but did not detect or cross-react with DNAfrom C. albicans or any other Aspergillus species tested. FIGS. 4-5 showthe A. fumigatus real-time PCR assays based on SWI5 regions 1 and 3 andthe specificity of the assays for A. fumigatus.

Example 2

Additional primers to amplify A. nidulans, A. niger and A. terreus weredesigned. These primers produced PCR products from these species whichwere sequenced. The primer sequences are outlined in Table 7. AnigSWI5primer set were designed to amplify positions 43 to 512 to produce a PCRproduct of 469 bp in length, AterrSWI5 primer set amplified positions 44to 450 producing a PCR product of 469 bp and AnidSWI5 primer amplifiedpositions 40 to 510 creating PCR products of 406 bp.

Thirty strains representing 8 Aspergillus species (Table 8) have beensuccessfully sequenced with four different primer sets.

TABLE 7 PCR primers designed to amplify SWI5 gene regionsin Aspergillus spp. SEQ ID NO. 36 AnigSWI5_1F CAACACAGGCGGCSEQ ID NO. 37 AnigSWI5_1R TCTGTTGTTGTTGCATC SEQ ID NO. 38 AterrSWI5_1FAACATCGAAGGCAGA SEQ ID NO. 39 AterrSWI5_3R CTGCATCATGTTGAGGSEQ ID NO. 40 AnidSWI5_1F CGTCAACATCGACG SEQ ID NO. 41 AnidSWI5_1RTGCTGTTGAATGAGATT

TABLE 8 Initial panel used to generate SWI5 sequences Number Speciesname of strains Strain numbers A. fumigatus 5 505 + 359 + 2010 + 4185 +419 A. flavus 3 2008 + 117.62 + 110.55 A. niger 5 5184 + 329399 + 2864 +554 + 2828 + 2599 + 121 A. terreus 3 383 + 5677 + 307 A. candidus 0 — A.clavatus 3 5138 + 1348 + 7944 A. glaucus 2 117314 + 542 A. nidulans 4589 + 7063 + 808 + 670 A. versicolor 0 — N. fischeri 3 19912 + 1085 +241525

Thirty sequences representing 8 species of Aspergillus were generated.These sequences are listed in Appendix 1. Alignments were produced usingthe Clustal W software and homology and sequence differences wereidentified (FIG. 11).

Results

Primer and Probe Design:

The sequence information generated was aligned using Clustal W.Potential primers and probes for real-time PCR assays were designed toamplify and detect A. fumigatus, A. flavus, A. niger and A. terreus.These primers and probes are outlined in Table 9. These assays wereevaluated on the LC480.

The assays which included the probes Afum_SWI5_1, Aflav_SWI5_1,Anig_SWI5_1 and Aterr_SWI5_1 proved to be specific, under thermocyclingconditions which included annealing at 95° C. for 10 seconds and 60° C.for 30 seconds for 50 cycles (FIG. 6). The species tested in the assayswere A. fumigatus A. flavus A. niger A. terreus A. candidus A. clavatusA. glaucus A. nidulans A. versicolor N. fischeri.

To investigate the LOD of these assays cycling conditions of 95° C. for5 seconds and 60° C. for 10 seconds for 50 cycles were tested. This wasdone in an effort to reduce the overall assay time. A LOD of 10 cellequivalents was obtained for the A. fumigatus, Afum_SWI5 assay (FIG. 7).However, the other three assays did not perform as well. The A. terreus,Aterr_SWI5_1 assay produced a LOD of 10⁵ cell equivalents. (FIG. 8). A.niger and A. flavus assays did not produce a LOD. (Data not shown).

To improve the assays, new primers and probes (Table 9) were designedfor the detection of the SWI5 target in the species of interest. Thedetection limit for the new A. fumigatus assay Afum_SWI5_2 was found tobe 2.5 cell equivalents per reaction under thermocycling conditionswhich included annealing at 95° C. for 10 seconds and 60° C. for 30seconds for 50 cycles. (FIG. 9a ).

When the annealing times of the Afum_SWI5_2 assay were reduced, an LODof 100 cells per reaction (FIG. 9b ) was obtained. The A. niger assayAnig_SWI5_2 showed potential with a detection limit of 5 cells perreaction (FIG. 10).

TABLE 9 Probes and primers for real-time PCR assays forthe detection of the SWI5 target. Oligo name Sequence 5′-3′SEQ ID NO. 42 Afum_SWI5_1 ccgtctttgacctcagaaaga SEQ ID NO. 43Afum_SWI5_1F cccaattctcgcaat SEQ ID NO. 44 Afum_SWI5_1R ccggatgattcgcaSEQ ID NO. 45 Aflav_SWI5_1 tgcaacagcaacactatgct SEQ ID NO. 46Aflav_SWI5_1F agaccgtgcaagat SEQ ID NO. 47 Aflav_SWI5_1Rggtttgcaattcttca SEQ ID NO. 48 Anig_SWI5_1 cctgtgtgtagcgcagcSEQ ID NO. 49 Anig_SWI5_1F acagtaccgcagc SEQ ID NO. 50 Anig_SWI5_1Rcttccggggtgaa SEQ ID NO. 51 Aterr_SWI5_1 cgatctctacatgttcaacgacSEQ ID NO. 52 Aterr_SWI5_1F cgaaagctccct SEQ ID NO. 53 Aterr_SWI5_1Rccgtctgcggtc SEQ ID NO. 54 Afum_SWI5_2 attccttgctggaggagaacaSEQ ID NO. 55 Afum_SWI5_2F gcacgatgggaccgt SEQ ID NO. 56 Afum_SWI5_2Rgattgcgagaattggg SEQ ID NO. 57 Afum_SWI5_3R ccgattgcgagaattgggSEQ ID NO. 58 Afum_SWI5_3F acgatgggaccgt SEQ ID NO. 59 Aflav_SWI5_2Fgtctctacacatgcaacgatcgc SEQ ID NO. 60 Aflav_SWI5_2Rtgcattaccaggggacctgtt SEQ ID NO. 61 Aflav_SWI5_2tgtgcaagatggtaaccttctaaat SEQ ID NO. 62 Aterr_SWI5_2Fgaccatgcacgatggtaacgtt SEQ ID NO. 63 Aterr_SWI5_2R gtgggtccgaaacgttgcaSEQ ID NO. 64 Aterr_SWI5_2 agacggtgatgggagaagt SEQ ID NO. 65Anig_SWI5_2F atccatgcaagatggtac SEQ ID NO. 66 Anig_SWI5_2Rctacacacaggtatcgtt SEQ ID NO. 67 Anig_SWI5_2 tgctcggggacagcca

TABLE 10 Thermocycling conditions LC480 thermocycling conditions StepTemp Time UNG 50° C. 2 min 40-50 cycles Denaturation 95° C. 1 minCycling 95° C. 5-10 secs 60° C. 10-30 secs Cooling 40° C. 1-2 mins

TABLE 11 Initial exclusivity panel for the SWI5 assays Species name A.fumigatus 2078 A. flavus 117 A. niger 2599 A. terreus 2729 A. candidus567.65 A. clavatus 2391 A. glaucus 117314 A. nidulans 7063 A. versicolor2916 N. fischeri 214525Discussion

The number of yeast and fungal infections among immunocomprised patientsis escalating. Contributing to this increase is the growing resistanceof many yeast and fungal species to antifungal drugs. There is thereforea need to develop a fast, accurate diagnostic method to enable earlydiagnosis of fungal and yeast species. Early diagnosis will enable theselection of a specific narrow spectrum antibiotic or antifungal totreat the infection. The current invention provides for sequences and/ordiagnostic assays to detect and identify one or more fungal and yeastspecies. The current inventors have exploited the sequence of the SWI5gene in Aspergillus species to design primers and probes specific forregions of this gene. The SWI5 gene encodes a zinc finger DNA-bindingprotein required transcriptional activation of genes expressed inG1-phase and at the G1/M boundary. The sequence is conserved amongclosely related yeast and fungal species. The SWI5 sequence hassignificant intragenic sequence heterogeneity in some regions, whilehaving significant homogeneity in others, a trait which makes SWI5 anideal candidate for the design of primers and probes directed towardsthe detection of yeast and fungal species specific targets and for thedetection of genus specific diagnostic targets respectively. The currentinvention allows the detection of yeast and fungal species.

The words “comprises/comprising” and the words “having/including” whenused herein with reference to the present invention are used to specifythe presence of stated features, integers, steps or components but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The invention provides sequences and/or diagnostic assays to detect andidentify one or more yeast or fungal species. The current inventors haveused the SWI5 gene sequence to design primers and probes that arespecific to Aspergillus and Candida glabrata SWI5 polynucleotidesequences. Such primers not only allow the detection of yeast and fungalspecies but also allow identification of Aspergillus species anddiscrimination between Aspergillus species and Candida glabrata. Thecurrent invention further provides for primers and probes that allowidentification of Aspergillus species and Candida glabrata.

All patents, patent applications, publications, and accession numberscited herein are incorporated by reference in their entireties.

In so far as any sequence disclosed herein differs from its counterpartin the attached sequence listing in PatentIn3.3 software, the sequenceswithin this body of text are to be considered as the correct version.

SEQ IDs

N or x=any nucleotide; w=a/t, m=a/c, r=a/g, k=g/t, s=c/g, y=c/t,h=a/t/c, v=a/g/c, d=a/g/t, b=g/t/c. In some cases, specific degeneracyoptions are indicated in parenthesis: e.g.: (a/g) is either A or G.

SEQ ID NO 1: AspSWI5-1-F ATCGACAACATCGTCGGCAGA SEQ ID NO 2: AspSWI5-1-RGCTGTTGCTGTTGCATCAGATT SEQ ID NO 3: AspSWI5-2-F TAGCCGCCATGCCAAGCSEQ ID NO 4: AspSWI5-2-R CCAGTCTCTTTGATAGAAGCA SEQ ID NO 5: AspSWI5-3-FCGTGGACATGACCTG AAG C SEQ ID NO 6: AspSWI5-3-R GTCTCTCCTCCAACTCTGGSEQ ID NO 7: 1F ATGTTAGCCAATCCAC SEQ NO 8: 1R ATTCCAGGCACCG SEQ NO 9: 2FCTTGAGGGCCAAATC SEQ NO 10: 2R CTCGTCCTTTCAATCC SEQ NO 11: 3FACTATGCCTCGTCG SEQ NO 12: 3R AGCGAATACATTGCC SEQ NO 13: 4F:ACAAACCATATGAATGTC SEQ NO 14: 4R GCAGGCTCGGTT SEQ NO 15: 5FCCTCGAGAAGATCGT SEQ NO 16: 5R CTAGCAGTCCATGAAGSEQ ID NO 17: P1-AspSWI5-1 CCAAAGTTCCTACCCTTCCAGCACSEQ ID NO 18: P1-AspSWI5-3 CTGACTCGGCACAGACAACGAGGA SEQ ID NO 19:>AF419.64-SWI51\(AspSWI5-1F) SWI5 sequences generated for A. fumigatusTTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCATTGCTATGGGAAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACAGCSEQ ID NO 20:>NF1085-SWI51\(AspSWI5-1F) SWI5 sequences generated for N. fischeriTCTCCACTCCCAATGCGCTGGAGGCCGCCAAAGTTCCTACACTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGGCAGAGTCTCGACCAACGTCCTCGACCTATGCACGATGGGGCCGTTTCCATTACTAACGCAACAGCAACTCAGCAGTCCCGAATCCTTGCCGGGGGAGCGCATCATCCCCGATTCTCGCAATCGGCGCATTTCCCTCAGCATTCCTCTCCCATGCCTGTGATGCCTGAATGCCCGTCTCTGACCTCGGAAGACTTGGAAGCACTATCCAATTCTACCAGCAACGCGAACGATCCAGGCATGGCTTACATGAATTCGAGCTTCATTCCCATGGGAAACCCGAGCTTGGGGAATCGACCAATGGACAGCAATCTCAATCTGATGCAACAGCAACAGCSEQ ID NO 21:>AN670.78A-SWI51\(AspSWI5-1F) SWI5 sequences generated for A. nidulansTCTCGACTCCCTCCGCGCTCGATGCCGCAAAACCTCCAGGCCTTTCTCCACAGGCTCTGCAGAGATACCATGCTCATCGCCGCGGCCAAAGTCTGGACCAGCGAGCTGTGCAAGCTCAAGCTCAGCGACAACAGCTCGTGCAAGATGCGTCAAGTACTAACCAAACAGCACCCCAATTCGCGCCTAACTCAACCCTCGTCCCTTTAATGCCTGACTCCCAGATCTTCGGCCAAGACGACATGCAGGCTTCAAGTCACGCCAATTACCAGACGCCTCACAGTCTACCCTACTTGCACACGAACTTTGTCAAGGCCGATGATCAGGCTCGGGATGCTCGACCTGTCAATCACCACCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 22:>AC5138-SWI51\(AspSWI5-1F) SWI5 sequences generated for A. clavatusTCTCAACCCCCAATGTCCTGGAAGCCGTCAAAGTACCTACTCTCCCGGCGCAGGCTTTGCAGCGCATCCAGGCGCATCGTCGGGGACAGAGTCTCGATCAGCGATCTGTGCATGCCCAACGATCTCGTCCCATGCAAGATGGTGGTCCTTCCATTACTAACCCAGCAGTGCCTCAGCAACCCCAGATGGTTGCCGGGGGAGCGCCTCATCAGCAATTCCCTCAATCGTCGCAATTCCCCCAGCAACTTACCCCCATGCCTATGATGCCCGAATGTCAGTCGTTTCCCTCCGACGAGTTGCAGGCGTTGTCCGGACAGAGCATCAACGTGAATCAACCGGACATGGCTTATATGATTCCAGACTTCGTCAACATCGGAAATCATTGCGTTGGGAACCGACCCATGGTCAGCAACCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 23:>AN329399-SWI51\(AspSWI5-1F) SWI5 sequences generated for A. nigerTCTCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGCTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAATCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCATGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGGACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCAATCTGATGCAACAGCAACAGC SEQ ID NO 24:>AF2008-SWI51\(AspSWI5-1F) SWI5 sequences generated for A. flavusTTTCAACACCTTCCGCTCTTGATGCCGCCAAAGTCCCCAGTCTTCCTGCCCAGGCAATGCACCGATACCATGCTCACCGTCGCGGCCAGAGCCTAGACCAGAGGTCTCTACACATGCAACGATCGCAGACCGTGCAAGATGGTAACCTTCTAAATACTAACGCAACAGGTCCCCTGGTAATGCAACAGCAACACTATGCTCGTTCGGCGCAACCGACACCCATGCCCATGATGCCTGAGTGCCAGACTTTCAGTCCTGAAGAATTGCAAACCCAACCAAGTATGGGATACATGAGCCCAGCCTTCGCCAAGGCCGAGACCCCGGCGCTGGAGAGTCGGCCGATGAACCTCCATCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 25:>AF416.64-SWI53\(AspSWI5-3F) SWI5 sequences generated for A. fumigatusGTCACGCCAAGATTCACACTGGAGACAAACCATATGAATGTCTTTGTGGCAATGTATTCGCTAGGCACGATGCTCTGACTCGGCACAGACAACGAGGAATGTGCATCGGCGGTTACAAGGGTATTGTGCGCAAGACAACCAAGCGTGGCCGTCCAAAGAAACATCGACCAGAGTTGGAGGAGAGAC SEQ ID NO 26:>NF1085-SWI53\(AspSWI5-3F) SWI5 sequences generated for N. fischeriGTCACGCCAAGATTCACACTGGAGACAAGCCATATGAATGTCTCTGTGGCAACGTATTCGCTAGGCACGATGCTCTGACTCGGCACAGGCAACGAGGAATGTGCATCGGCGGTTACAAGGGTATTGTGCGCAAGACAACAAAACGTGGTCGTCCGAAGAAACATCGACCAGAGTTGGAGGAGAGAC SEQ ID NO 27:>AN670.78-SWI53\(AspSWI5-3F) SWI5 sequences generated for A. nidulansGCCACGCGAAGATCCACACAGGAGACAAGCCGTACGAATGTCTATGCGGTAATGTTTTTGCCCGGCACGATGCCCTAACTCGACACCGCCAGAGGGGAATGTGCATTGGTGGTTACAAGGGAATTGTGCGTAAGACAACGAAACGTGGCCGTCCTAAGAAGCACCGCCCATAGTTGGAGGAGAGAC SEQ ID NO 28:>AN1329399-SWI53\(AspSWI5-3F) SWI5 sequences generated for A. nigerGTCATGCTAAGATCCATACCGGCGACAAGCCTTACGAGTGCCTCTGTGGAAATGTATTTGCGAGACACGACGCCTTGACTCGACACAGACAGCGGGGTATGTGCATTGGCGGCTACAAGGGAATTGTGCGCAAGACAACGAAACGGGGTCGTCCGAAGAAGCACCGACCAGAGTTGGAGGAGAGAC SEQ ID NO 29:>A. fumigatus gi|70999549|ref|XM_749401.1| Aspergillus fumigatus Af293C2H2 transcription factor (Swi5), putative (AFUA_3G11250) mRNA,complete cdsATGTTAGCCAATCCACATAGTAATCTGCACGAGCGTTATCGACAACATCGTCGGCAGATTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCTTTGCTATGGGGAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACAGCAGCAAAATGCCCACGTCTCATGCGTCAATAGCCTTGAGGGCCAAATCCTCGACAACGGTGCCTGGAATTTCTACCAGCAAGGCCAGCTCCCTACGACGCTTCGGTCTCAAGTCAACAATCTTTCGGCCGATGGGAGACGACAGTCTGTTCAGTCAGATATCACCCCCTCGCAACGACCACATACCCCCAAGCAAGCAAATACGCACTACTTTCCCATAACGCCAGCGACAACTCCGTTTAAGAAACCGGCGGAACTTGCTCAGTATAGTACGGACATGCAGTCCACCCCCACCAAGGAGACAGGCCGCTCTGCACCCGCATCAGCCCAGTCGGTATACATGCAACGAGCCAAATCCCTCCAAGGAGTTGCGGGGTCTACCTTTTCCAACTCCAAAATCGAGATGCCCTCCCCCCCGAACACGGCTTCATTTGAAATTGACAATTTTGATGCGTTTAGCAGTCAGCAGGGTTCCAGTTTCGAGATTTCCGAGTCAGAGAATTTATCGCAGAGTCACTATGCCTCGTCGTCAGCAACCTCGTCCTTTCAATCCTCCCCAGAGCTAGCCGCCATGCCAAGCCCCGAAGACAATCACGAGAAGGCTCATAAGCTGCCCATCTTCCCTGCCGCGTCCAATCGGCCAGCGCACAGGAAGGCGCTGAGTACCAGCTCCAGTTCCACCTTGACGAAACCCCGGCTCTCTCCAAGAGTGGCTTCAATTGATAGCCTTAACCTTGATGCTAGGGTTCATGCTTCTATCAAAGAGACTGGGATCACCATCGATGAGATCGCCTCCTACATCTCTGGCCCTGACCCGGAGGATGGAAAGTGGGTTTGCCTTCACCCCGGCTGCGAGCGGCGGTTCGGAAGAAAAGAAAATATCAAGTCCCACGTCCAAACCCACCTTGGTGATCGCCAATATAAGTGTGATCATTGCAACAAGTGCTTCGTTCGTGGACATGACCTGAAGCGTCACGCCAAGATTCACACTGGAGACAAACCATATGAATGTCTTTGTGGCAATGTATTCGCTAGGCACGATGCTCTGACTCGGCACAGACAACGAGGAATGTGCATCGGCGGTTACAAGGGTATTGTGCGCAAGACAACCAAGCGTGGCCGTCCAAAGAAACATCGACCAGAGTTGGAGGAGAGACAGGATAAGGCAGCCAAGACACGCCAGAGAGTCGCCGAAAAGTCATCCCATGACTCCTCGTCCGGGTGTGTTGATTCCCCCAACTCGCCGCCTTCCGAAATCCTTGAAAATATGAGTCTTCACGGGGGATCGAGCCCTAAAGAGAATATGCCCGCGTTCATTCAGCCCAACTTTTCCTTGCCTCCATCGGCGTTTACTTTCACGCCTCCTGCGTCTCCCCGACAGAGTCTTGGAAACCAGCCATCGCCCGCTCAGAGTCGCCGCTCACTCACGCCCAGTAGCGAGGATGAAATGCTGCCTTTGTCTCCCTCCAAGCGCCCCCTCGAGAAGATCGTTGAAGAACCGAGCCTGCCTTTCACTTCGAGTGCCGACCCATACACCGATATTGCTGCCTCCACCGCGGAGCTGTCTTCTCCACATACGGCTCCCACCTTGGCTGATTCGTCTCACGGCTCCGACCTCGATATTTTCATCAGCACGGATAGCTCCGCCAATTTCAAGCATGAATTTCCCGATCTGAGTGACCCCGACATGGCCGCTTTCCCCGACTATGTCAATGGGTCTACCTTCGAACCCGGAATGGATCTGTTCTCGAGCAAGACATTCTCTGCCGGTACCTCGATGAACGAGGACTTCTTTTCACTCCAATTCCAGGTTGATGATATGACCAAAGAATTCTTCATGGACTGCTAG SEQ ID NO 30: >N. fischeri gi|119491684|ref|XM_001263336.1|Neosartorya fischeri NRRL181 C2H2 transcription factor (Swi5), putative (NFIA_066040) mRNA,complete cdsATGTTAGCCAATCCACATAGCAATCTGCACGAGCGTTATCGACAACATCGTCGGCAGATCTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGTCCAACGTCCTCGACCTATGCACGATGGGGCCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCCGGGGGAGCGCAGCATCCCCGATTCTCGCAATCGGCGCATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCGGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAACCATCCGGGCATGGCTTACATGAATTCGAGCTTCATTACCATGGGAAATCCGAGCTTGGGGATTCGACCAATGGATAACAATCTAAATCTGATGCAACAGCAGCAGCTGCAAAATGCTCACGTCTCATGCGTCAATAGCCTTGATGGCCAAATCCTCGACAACGGTGCCTGGAATTTCTACCAGCAAGGCCAGCTCCCTACGACGCTTCGGTCTCAAGTCAACAACCTTTCGACCGATGGGAGACGACAGTCTGTTCAGTCAGATATCACCCCCTCGCAACGACCGCATACCCCCAAGCAGGCAAATACGCGTAAGTTATCCGTTCAGTCATACTGGTCATTTTTCTCCCAGCCACTAACGACCGATCCAGACTACTTTCCCATAACGCCAGCGACAACTCCGTTCAAGAAACCAGCGGAACTTGCTCAGTATAGTACGGACATGCAGTCCACCCCCTCCAAGGAGACAGGCCGCTCTGCACCCGCATCAGCCCAGTCGGCATACATGCAACGAGCCAAATCCCTCCAAGGAGTTGCCGGGTCTACCTTTTCCAACTCCAAAATCGAGATGCCCTCCCCCCCGAACACTGCTTCATTTGAAATTGACAATTTTGATGCGTTTAGCAGTCAGCAGGGTTCCAGTTTCGAAATTTCCGAGTCAGAGAATTTATCGCAGAGTCACTATGCCTCGTCGTCAGCAACCTCGTCCTTTCAATCCTCCCCAGAGCTAGCCGCCATGCCAAGCCCAGAAGACAATCATGAGAAGGCTCATAAACTGCCCATCTTCCCTGCCGCGTCGAGTCGGGCAACCCACAGGAAGGCGCTGAGTACCAGCTCCAGTTCCTCCTTGACGAAACCCCGGCTCTCTCCAAGAGTGGCTTCAATTGATAGCCTTAACCTTGATGCTAGGGTTCATGCTTCTATCAAAGAGACTGGGATCACCATCGATGAGATAGCCTCCTACATCTCTGGCCCCGACCCGGAGGATGGAAAGTGGGTTTGCCTTCACCCCGGCTGCGAACGGCGGTTCGGAAGAAAAGAGAATATTAAGTCACACGTCCAAACCCACCTTGGTGATCGCCAATATAAATGTGATCATTGCAACAAGTGCTTCGTTCGTGGACATGACCTGAAGCGTCACGCCAAGATTCACACTGGAGACAAACCATATGAATGTCTTTGTGGCAACGTATTCGCTAGGCACGATGCTCTGACTCGGCACAGACAACGAGGAATGTGCATCGGCGGTTACAAGGGTATTGTGCGCAAGACAACAAAACGTGGTCGTCCAAAGAAACATCGACCAGAGTTGGAGGAGAGACAGGACAAGGCAGCCAAGACACGCCAGAGAGTCGCCGGGAAGTCATCCCTTGACTCCTCGTCCGGGTGTGTTGATACCCCCAACTCGCCGCCTTCCGAAATCCTTGAGAATATGAGTCTTCACGGTGGATCGAGCCCCAAAGAGGATATGCCCGTGTTCATCCAACCCAACTTTTCCTTGCCTCCATCGGCGTTTACTTTCACGCCTCCTGCGTCTCCGCGACAGAGTCTTGGAAACCAGCCATCGCCCGCTCAGAGTCGCCGCTCACTCACACCCAGTAGCGAGGATGAAATGCTGCCTTTGTCGCCCTCCAAGCGCCCCCTCGAGAAGATTGTTGAAGAACCGAGCCTGCCTTTCATTTCGAATGCCGACCCATATACCGATATTGCTACCTCCACCGCCGAGCTGTCTTCTCCACATACGGCTCCCACCTTGGCTGATTCGTCTCACGGCTCCGACCTCGATATCTTCATCAGCACGGACAGCTCCGCCAACTTCAAGCATGAATTTCCCGATCTGAGTGACCCCGACATGGCCGCTTTCCCCGACTATGTCAATGGGTCTACTTTCGAGCCCGGACTGGATCTGTTCTCGAGCAAGACATTCTCTGCCGGTACCTCGATGAACGAGGACTTCTTTTCACTCCAATTCCAGGTTGATGATATGACCAAAGAATTCTTCATGGACTGCTAG SEQ ID NO 31:>A. clavatus gi|121705723|ref|XM_001271124.1| Aspergillus clavatus NRRL1 C2H2 transcription factor (Swi5), putative (ACLA_039140) mRNA,complete cdsATGTTTGCCAATCCGCACAGTAACCTTCACGAGCGGTATCGACAACATCGCCGGCAGATCTCAACCCCCAATGTCCTGGAAGCCGTCAAAGTACCTACTCTCCCGGCGCAGGCTTTGCAGCGCATCCAGGCGCATCGTCGGGGACAGAGTCTCGATCAGCGATCTGTGCATGCCCAACGATCTCGTCCCATGCAAGATGGTGGTCCTTCCATTACTAACCCAGCAGTGCCTCAGCAACCCCAGATGGTTGCCGGGGGAGCGCCTCATCAGCAATTCCCTCAATCGTCGCAATTCCCCCAGCAACTTACCCCCATGCCTATGATGCCCGAATGTCAGTCGTTTCCCTCCGACGAGTTGCAGGCGTTGTCCGGACAGAGCATCAACGTGAATCAACCGGACATGGCTTATATGATTCCAGACTTCGTCAACATCGGAAATCATTGCGTTGGGAACCGACCCATGGTCAGCAACCTCAATCTGATTCAACAGCAGCAACTGCACAATCCCCATATCATTGCAAACAGTGCGCTCGATGGCCAGATTCTCGACAACAGCGCTTTCAATATCTATCAGCATGGCCTTCGACCCCAGACAAACAATCTTTCAGTGGATACACGACGATTGTCAGTTCACTCGGATGTAAGCCCCTCGCATCAGCCACATACGCCCAAGCAGACGAATTCGCGTAAGTCCACTGTCTCTCCATGGCGAATCCTGAGTCCATCAACTCCCGACTCACGACTGGAATCAGAATATTTCCCGATTACCCCAGCAACAACTCCTTTCAAGAAAACAGCCGAACTTGCTCAGTATAGCACGGACGTCCAGACAACTCCCTCCAAGGAGCAACGCTTTTCGGCCGCTCAGGCGGCCTACATGCAGCGGGCCAAGTCCCTTCAGGGCGTGGCCGGAACTACCTTTTCTCAACCAAAGATCGAGATGCTTTCCCCCCATAACACAGGTTCGTTTGAAATTGAGAGTTTTAATACTTTTGGCAGTCAGCAGGGTTCCACTTTTGAATTTTCCGAGTCAGAGAATTTGTCGCAAGGCCAGTATGCCTCGTCGTCAGCAACGTCATCCTTCCAATCCTCCCCAGAGCTAGCGGCCATGCCAAGCCCCGAGGACCATAACGAAAAGGCGCACAAGATCCCCATCTTCCCAGCCGTATCCAGCCGTATCAGTCACAAGAAGACTTTGAGTCTTCCCGCTAGCACTTCGCCGGCGAAACCCAAGCTTTCTCCCAGAGTGGCGTCCATAGACAACCTGAACCTCGATGCCCGTGTGCACGCCTCAATCAAAGAGACCGGTGTCACCATTGACGAGATTGCTTCCTACATCTCCGGCCCGGATCCAGAGGATGGAAAGTGGGTTTGCATTCACCCTGGTTGCGAGCGGCGGTTTGGAAGAAAAGAAAACATCAAGTCACACGTCCAAACACATCTGGGAGATCGCCAGTATAAATGTGACCATTGCAACAAGTGTTTCGTCCGCGGACATGACCTGAAGCGTCATGCTAAGATCCACACTGGAGACAAACCGTACGAATGCCTTTGCGGGAACGTTTTCGCTAGACACGATGCTTTGACCCGCCACCGACAGCGAGGCATGTGCATCGGTGGTTACAAGGGAATTGTGCGCAAGACAACAAAACGCGGTCGTCCTAAGAAACACCGGCCGGAGATGGATGAAAGGCAGGACAAAGCCGCCAAAACACGCCAGCGAGTCGCTGATAAGACATCCTTTGACTCCTTGTCTGGGACAGATGTTGCGCCGAATTCACCACCATCCGAAGTTCTTGAGAACATGAGCCTACACGGGGATCCAAGCCCAAAAGAAGAGATGCCCGCGTTCAACCAGCCCGATTACTCGTTACCACCCTCTGTTTTCACCTTCACGCCTCCTGCATCGCCAGGGCACAACCTTGGAAACCGGCCATCACCGAATCAGAGTTACCGGTCTCTCACGCCCAGTAGTGAAGATGAAATGCTGCCTTCGTCGCCTATCAAGCGGCCTCTAGAGAGGATCGCCGAAGAGTCGGGGTTGCCTTATATCGAACATGCAGATCTCTATACTGAGATCGCCACTTCTGCTGCTGATCTGTCGTCTCCACACACCGCTCCTACCTTGGCCGATTCATGTCACGGCTCTGATCTCGATATCTTCATCAGCCCTGACAGCTCTGCGAACTTCAAACATGAATTCCCTGAGCTGAGTGACATGGCCGCTTTCCCTGACTATACGAATACCTCTACCTTCGACGCCGGACTGGACCTCTTCTCAAGCAAGAACTTCTCCACTGTTCCTTCAATGAATGACGATTTCTTCTCCTTCCAATTCCAGGCTGACGACCAACCCTTGGATGTCATGGCCAAGGAGTTCTTCGCCGACTGA SEQ ID NO 32: >A. terreus gi|115396393|ref|XM_001213836.1|Aspergillus terreusNIH2624 conserved hypothetical protein (ATEG_04658) mRNA, complete cdsATGCTATCCACCCAGCACCGAAATTCGCATGACCGGCACCGGCAACATCGAAGGCAGATTTCCACCCCTTCTGCCATTGACGCCGCGAAAGCTCCCTCCCTGCCGGCGCAGGCATTGCATCGATACCATGCTCATCGCCGAGGCCAGAGTTTCGACCAACGATCTCTACATGTTCAACGACCGCAGACCATGCACGATGGTAACGTTTCAGCTACTAACAACACAGGACCGCAGACGGTGATGGGAGAAGTGCAGCAGCTGCAACGTTTCGGACCCACAGGACACTCAGGCTATCACCAACACTCGGCTTCTATGCCGGTAATGCCCGAGTGCCAACCATTGAGCCAGGAAGACTTCCAGACCCTGGGCAATCGCAATGTCCCAGACAACCAGACCGCCATGACCTATATGACGCCCGCGCTGCACCTCAACATGATGCAGCAGCAGCAGCAGCTTCAACACGCAAGAGTGCATTCCAACAATGCGCTTGATGGTCAGCTTCTCGAGAATGGTCCGTGGGACATGTATCAGCACGACAACCTTGCTGCGCCGCTCCCACAGCAACCCAACACCATTCCCGCGGGCTTAAGGCGTCTGTCAGCTCAATCAGAGACCACTCCTGCGCAACGACCACTCACTCCGAAGCATAACAATACCAATTACCTTCCTATCACCCCTGCCACAACGCCCTTTAAAAAATCAGTGGATCTTGCCCAGTACAGCGGCGAACTCCATTCAACTCCCACCAAGGACCAGAGCCTTTCCGCACCCGGCTCTTCCCAATCGTTCATGCAACGTACGAATTCACTCCAAGGAGTGGCTGGAACAACATTCTCCCAACCCAAGCTTGAAGTCCCCTCCCCCCCAAACACTGCGTCATTTGATGTGGACAGCTTCGATGCTTTTGACTATCAACAGGAATCCAGTTATGAAATCCCCAAGTCTGAGAGCCTCAACCATTATGCCTCGTCGTTGGCGTCGTCATCATTCAACTCATCCCCGGAACTCGCGGCTATGCCGTGTCCACAAGACGGCGGTAGAGCGCAAAAGCTCCCCATCTACCCGGTCACACCAAGTCGCACAAACATGAAGAAGTCTCCCAGCGTCACCTCCAACTCGTCCGCGTCGAAGCCAAAGCTCTCTCCAAGGGTCGCAACCATTGACAGCCTCAACCTGGATGCCAGAGTCCATGCATCCATCAAAGAAACTGGCGTCACAATCGACGAGATTGCCTCGTACATAAGCGGCCCTGATCCAGAGGACGGAAAATGGGTGTGTCTGCACCCAGGATGCGAGCGTCGGTTTGGAAGAAAAGAAAACATCAAGTCCCATGTTCAGACCCATCTGGGTGACCGTCAGTACAAGTGCGACCACTGCAACAAATGTTTTGTCCGCGGCCACGATCTCAAACGCCATGCCAAAATCCATACCGGTGACAAGCCGTACGAGTGTCTCTGCGGTAATGTGTTCGCTCGACACGACGCATTGACGCGCCACCGACAACGGGGCATGTGCATCGGTGGTTACAAGGGAATCGTTCGCAAGACAACGAAACGTGGTCGCCCCAAGAAACATCGGCCCGAGATGGACGAGAGACAAGATAAGGCATCTAGAACCCGCCAGCGGCTCGCCGAGAAGACATCTTTCGACTCCTCCAGCTCAGATATCTCTCGCAATTCTCCCCCATCGGAGGTACTGGAACAAATGAGCCTTCACGGCTCTAGCCCCGCCGAACAGATGCCGGTATTCCATAATCCAAACTACTCGCTGCCTCCGGAGGTCTTTACGTTCACTCCTCCTGCATCCCCTGGCGGTAGCGCTGGAAACAACCCTTCTCCAAGCCACAGCCAACGCTCCCTCACGCCTAGCACCGAGGACGAAATGCCACCTTTGTCACCTTCCAAGCAACCTCTGTCAAAGATTGTGGAAGAATCTGGCTTGCCTCTTATGCCCGATTGTGCATACACCAATGCCACCAACTCAACCATCAATGCTTTGTCGTCCCCGCATACCGCACCCACCCTGAGTGATGCTTCAAACGGCTCCGATCTTGACATCTTTATCAGCCAAGATCCGTCCACCGGCTTCGGCAAGCATGAGTTTTCCGACCTCACTGATTCCGACATGGCGGCATTCCCTGACTATGTGAACGGCTCTTCCTTCGAAGGCGGAATGGACCTCTTCCAAGGAAAGGGGTTCTCCAATGCTCCCCCAATGAGCGACGACTTCTTCTCCTTCCAATTTCAAGTTGACGAGCAACCATCCGATGTTATGACCCGCGATTTCTTCATGGATTAA SEQ ID NO 33:>A. niger gi|145232922|ref|XM_001399797.1| Aspergillus niger CBS 513.88hypothetical protein (An02g07000) mRNA, complete cdsATGCTGTCCACGGCGCACAGCAACCTTCATGAGCGACATCGACAACACAGGCGGCAGATCTCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGTTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAATCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCGTGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGGACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCGACCTGATGCAACAACAACAGATGCACAGCACTAAGGTTCCGTGCGGGTCTGGAACGGAAGGTCATTTTTTCGAGACGGGACTTTGGGACTTTTACCAGCCAACTTTCCCCCCTCACGCTGATGTAAGGAAGTTATCGGTGCAGTCGGATGCTACTCCGTCTCAGCACCCGCATACGCCTAAGCACGGCAATACACAGTATGCCCCGATTACACCAGCAACAACACCATTCAAGCAAACCGTGGGCCTAGCTCAGTACGGTGGGGACATCCAGTCAGGATCCACCAAAGATCAGGGTAGCGCGATGCCCGGATCAGCCCAGTCGTCGTACATGCAGCGAGCAAAGTCTCTCCAAGGCGTGGCTGGAACTACTTTCACGCAGCAGAAGTTTGATGTTTCTACCCCCCCGAACACAGCATCATTTGAAGTGGATAACTTTGATACTTTTAACTATGAGCAGGGTTCTAGCTTTGAGGTTCCTAAATCGGAAAGTCTGTCACAAAGCCAGTATGCATCGTCGTCGTCGGCATCATCATCATCCTTCATGTCATCTCCCGAGCTTGCGGCTATGCCTTGCCCCGAAGATGGAGGTGCAAAGACCCCCAAAATCCCTATCTATCCCGCCACTCCCAGCCGTCCGCATCACAGAAAGACGCCCAGTGCAACACCTAGCTCATCGGCCAAGCCAAAGCTTTCTCCGCGCGTTGCGTCTATCGATAACCTGAACCTCGACGCTCGCGTGCAAGCATCAATCAAAGAAACGGGTGTCACCATTGACGAGATTGCTTCGTACATTCATGGGCCTGACCCGGAGGACGGGAAATGGGTATGCCTGCACCCCGGCTGCGAGCGCCGCTTTGGAAGAAAGGAGAATATCAAGTCACATGTCCAGACTCACCTGGGCGACCGCCAGTACAAGTGCGATCATTGCAACAAATGCTTTGTTCGCGGTCATGACCTTAAGCGTCATGCTAAGATCCATACCGGCGACAAGCCTTACGAGTGCCTCTGTGGAAATGTATTTGCGAGACACGACGCCTTGACTCGACACAGACAGCGGGGTATGTGCATTGGCGGCTACAAGGGCATTGTGCGCAAGACAACGAAACGGGGTCGTCCGAAGAAGCACCGACCAGAGATGGATGAGAGACAGGACAAAGCATCGAGAACGCGTCAGAGGATTGCAGAGAAGTCATCTTTCGACTCGTCCACATCCGAGTCCTCACGCAACACGCCTCCTTCCGAAGTCTTCGAAAACATGAGCCTGCATGGTTCTAGCCCGGCGGAAGAGATGCCAGTGTTCAACAACCCCAACTACTCGTTGCCACCAGAGGTTTTCACATTCACGCCTCCTGCATCTCCCGGTTACAGCGTGGGAATCAAGCCATCGCCTTCTCGGGACGAGCGATCGATCACCCCCAGCTCAGAAGATGAAATGCTTCCTTCCTCACCATCAAAGCAGCACCTCGAGAGCCTCGTCACAGACTCCAGCTTGCCTTACATGTCTGATCCGGAGACATGCCCGTATACAGATGCTTCCGGCGCTGCTAGCCATTCTCTATCTTCACCCCATGCCGCTCCCACCCTGTCCGAATCATCTAACGGCTCTGATCTCGACATTTTTATTAGCCAGGATTCGACCTCTGGTTTTGGAAAGCCCGAATTCGGAGACCTGGCTGATCCCGACATGGCCCCGTTCCCAGACTATGTGAACACGACGTCCTTTGAAGGTGGTCTGGAACTGTTCCCCAACAAGCCCTTCTCCTCGGGCCCCGTCATGGCCGACGACTTCTTCTTCCAATTTCAAGTGGACGAACAAGCCTCGGATGTTATGACTAAAGAATTCTTCATGGACTAA SEQ ID NO 34: >A. nidulans gi|67537405|ref|XM_657385.1|Aspergillus nidulans FGSC A4 hypothetical protein (AN4873.2), mRNAATGCTTTCTAATCCACAAAGTACCCTTCACGGGCGCCATCGTCAACATCGACGGCAGATCTCGACTCCCTCCGCGCTCGATGCCGTAAAACCCCCAGGCCTTTCTCCACAGGCTCTGCAGAGATATCATGCTCATCGCCGCGGCCAAAGTCTGGACCAGCGAGCTGTACAAGCTCAAGCTCAGCGACAACAGCTCGTGCAAGATGCGTCAAGTACTAACCAAACAGCACCGCAATTCGCGCCTAACTCAACCCTCGTCCCCTTAATTCCTGACTCCCAGATCTTCGGCCAAGACGACATGCAGGCTTCAAGTCACGCCAATTACCAGACGCCTCACAGCCTACCCTACTTGCACACGAATTTTGTCAAGGCCGATGATCAGGCTCGGGATGCTCGACCTGTCAATCACCATCTCAATCTCATTCAACAGCAGCAGCAACAACTGCACAATGCTAAGCTCAACTGCCACGATACACACGATGATCAGCTGCTCGACAACGACGCGTGGGATACATACAAACCCGACATCGCGTCCTCGCTTCAACAAACGACCACCGATATGAGACGACAATCTGTCCATTCAAACCCAAGTAGCTCATACCATCCGCACACTCCGAAAAAAACAAACTCACCAACGACGCCGTTCGACAAAACAGATTTTGCTCAGTACTGCGCGGAGACGCAAATCGTCCCAGCAAAAGACCAAAATGCTGCTGATGCCAGCTCCCAGTCGGCCTATATGCAACGCGCCAAGTCCCTTCAAGGAGTAGCGGGGACTAGCTTCTCACAGCAAAAGATTGAAATGCCCTCTCCCCCTAGCACTGATTCGTTTGCAGTTGATGGTTTTGATACGTTTGACTACCAGCAGTGTTCCAGTTTTGATAACCTCGCTACCACCAGCCACAGCCAGTACTCTACGTCGTCCAACTCACCAGAAGTCGCTGCCATTCCAAGCTCTGGAGATCACACCGAAAAGAAGTCCAAGCTCCCTATTTGTCCTGCCACGCCCAGCCGTCTCAGCCCAAGGAAACAGCTCGCTACGCCAAGCGCGGCTTCTTTAGTGAAGGCAAAACTTTCTCCGCGTGTCGCATCTATCGATAACCTCAACCTGGACTCCCGGGTGCATGCCTCTATCAAAGAAACTGGTGTTAGCATTGATGAAATAGCGTCCTATATCCACGGTCCAGACCCCGAAGACGGAAAGTGGGTGTGCCTGCACCCCGGCTGTGAGCGACGCTTTGGCCGCAAGGAAAACATCAAGTCACATGTGCAAACCCACCTAGGTGATCGCCAGTACAAGTGCGATCATTGTGATAAGTGTTTCGTTCGTGGGCATGATCTGAAGCGCCACGCGAAGATCCACACAGGAGACAAGCCGTACGAATGTCTATGCGGTAATGTTTTTGCCCGGCACGATGCCCTAACTCGGCACCGCCAGAGGGGAATGTGCATTGGTGGTTACAAGGGAATTGTGCGTAAGACAACGAAACGTGGCCGTCCTAAGAAGCACCGCCCAGAGATGGATGAGAGACGTGACAAGGCAACCAAGACCCGACAGAGGATCGCTGAGAAATCATTATTCAATTCTTCCGAATCGGACACTTCTCGTCGTACGCCGCCCTCGGAGGTGTTTGAGAACATGAGCCTTCATGGCTCCAGCTCAGCAGACGAGATGGTGACATTTGACAGCCAAAATTACTTGCCGCCAGAAGTGTTCACTTTCACTCCGCCCGAATCTCCAAATTACGGTACAGCAAGCAAGCCTGCCAGCCCGCGATCTCTCACGCCGAGCTCCGAAGACGAGATGCTACCTTTGTCATCATCCAAACGACCACTGGAAAACATTCTTGAGCATTCGGGCCTCCCCCTTCTCACTGATGCCGGCACATGCTCTTTCTCCTCTGTTTCAAGTTCAAGCAGCCATGCACTATCTTCTCCGCACACCGCGCCTACCCTAAGCGACCCTTCGCAACCATCCGATCTCGATATCTTCATCAACAGTGAACCTTCCTCTGCCTTTGGCAAACAAGATTTCGGCTTGGGTGATTCGGACATGGCTGCATTCCCAGACTACGTCAACGGCTCTGCGTTTGACAGCAGCTTGGATTTGCTCCAAGGGAAGAATTTCTCCACAGGGCCCTCTATGGGCGATGACTTCTTTTCCTTCCAGTTCCAAGTCGACGAACAAGCGTCGGACGTCATGTCAAGGGAGTTTTTCCTCGACTAASEQ ID NO 35 >gi|169770716|ref|XM—001819776.1|Aspergillus oryzea (AO090003000678) mRNA, complete cdsATGTTATCGAACCCACATCGCAATCTACAGGAACGACATCGACAACATCGGCGGCAGATTTCAACACCTTCCGCTCTTGATGCCGCCAAAGTCCCCAGTCTTCCTGCCCAGGCAATGCACCGATACCATGCTCACCGTCGCGGCCAGAGCCTAGACCAGAGGTCTCTACACATGCAACGATCGCAGACCGTGCAAGATGGTAACCTTCTAAATACTAACGCAACAGGTCCCCTGGTAATGCAACAGCAACACTATGCTCGTTCGGCGCAACCGACACCCATGCCCATGATGCCTGAGTGCCAGACTTTCAGTCCTGAAGAATTGCAAACCCAACCAAGTATGGGATACATGAGCCCAGCCTTCGCCAAGGCCGAGACCCCGGCGCTGGAGAGTCGGCCGATGAACCTCCATCTCAATCTGATTCAGCAACAGCAGTTGCAGCAAGCACAGCTCATGGAGAATGGCGCTTGGGATTTCTACCCACACGACAACCTCCCAACGGGACTTCCGCACCAGACCAACGCAATCCCTGCAGATATGAGACGACTATCAGTGCAGTCGGATGTCAGTCCCGCGCAAAGACCACATACGCCGAAACCTGCACGTAAGTGCCCTGATATTACTTACAGGAGACAAGCCCCACTGATGAAACTTAAAGACTACCTTCCCATTACCCCTGCGACAACACCATTCAAGAAAACAGTGGATCTTGTGCAGTATGGTGGCGACATGCAGCCAACCCCCACCAAGGAGCAGAGATTGTCTGTTCCCGTTTCAGCCCAGCCGTCGTACATGCAACGTGCTAAGTCTCTTCAAGGAGTGGCTGGGACGACCTTCTCCCAGCAAAAGATTGATATGCCCTCTCCCCCAAATACAGCATCCTTCGAGGTGGATAGTTTCGATGTGTTTAACTGCCAGCAGGGTTCCAGTTTTGAAATGTCAAAGTCTGAAAGTTTTTCATCTAGCCACTCTTCAACATCGTCGTCGTCAGCAACATCCCCTTTCAATTCGTCACCAGACCTTGCCTCCATGCCGCACCTTGCAGACAGTGGTAAGGCGCAGAAGATTCCTATTTACCCTGCAACACCTAGCCGTATGACTCCAAAGAAGACCCCAAGTGCGCCCCCGAGCTCGGCCAAACCCAAGCTTTCTCCAAGGGTAGCATCTATTGACAGCCTTAATCTTGACGCCCGGGTCCATGCCTCTATTAAAGAAACTGGTGTCACCATTGACGAGATAGCGTCATACATTCATGGCCCTGACCCAGAAGATGGAAAATGGGTATGCCTACACCCCGGTTGCGAACGCCGGTTCGGAAGGAAAGAGAACATCAAGTCCCATGTCCAAACACATCTTGGAGATCGCCAGTACAAGTGTGATCACTGCGATAAATGCTTCGTCCGCGGACACGACCTTAAGCGCCACGCCAAGATACATACCGGTGACAAACCATATGAATGCCTCTGTGGTAATGTGTTCGCCCGACATGATGCCTTGACTCGGCATCGGCAACGCGGCATGTGTATTGGCGGCTACAAGGGTATCGTGCGCAAGACCACCAAACGCGGTCGTCCGAGAAAGCACCGGCCTGAAATGGATGAAAGACAAGAGAAATCCTCCAGGACGCGCCAGAGAATCGCCGAAAAGTCGTCATTTGACTCTTCTGGATCAGACACTTCGCACAATTCGCCGCCCTCGGAAGTCTTCGAAAACATGAGCCTGCAGGGTTCTAGTCCGGTGGGAGAAATGCCAATGTTCAGCAATGTTAATTATTCATTGCCCCCTGAGGTCCTGACTTTCACACCTCCCGCCTCTCCTGGCGGTAGCATAAGAAACAGACCATCACCTGCCCACAGCCAGCGATCGATTACACCCAGCACTGAGGATGAAATGCCACCATTGTCTCCATCTAAACGACCTCTGGAAAGGATCATTGAAGAATCCGGTCTACCTTTAATTTCGGACCCTGAAGCCTGCCCCTACACAAACGCTACAAACTCAACAACTCATGCCCTATCTTCTCCACACACCGTGCCCACTTTGACCGAATCATCAAATGGCTCAGACCTAGACATCTTCATCAACCAAGATCCATCTACAAGCTTCAGCAAGCACGAGTTCCCTGGCTTAACCGACCCTGACATGGCGGCATTCCCTGATTACGTGAACGGTCCCGCTTTTGACAACGGCATGGATTTGTTTCAAAGCAAAGGTTTCTCTAACGGTCCCTCAATGAGTGACGATTTCTTTGCTTTCCAGTTCCAGATGGACGAACAACCATCGGACGTTATGACAAGGGAATTCTTCTTGGAGTGA SEQ ID NO 36: AnigSWI5_1F CAACACAGGCGGC SEQ ID NO 37: AnigSWI5_1RTCTGTTGTTGTTGCATC SEQ ID NO 38: AterrSWI5_1F AACATCGAAGGCAGASEQ ID NO 39: AterrSWI5_3R CTGCATCATGTTGAGG SEQ ID NO 40: AnidSWI5_1FCGTCAACATCGACG SEQ ID NO 41: AnidSWI5_1R TGCTGTTGAATGAGATTSEQ ID NO 42: Afum_SWI5_1 CCGTCTTTGACCTCAGAAAGASEQ ID NO 43: Afum_SWI5_1F CCCAATTCTCGCAA SEQ ID NO 44: Afum_SWI5_1RCCGGATGATTCGCA SEQ ID NO 45: Aflav_SWI5_1 TGCAACAGCAACACTATGCSEQ ID NO 46: Aflav_SWI5_1F AGACCGTGCAAGAT SEQ ID NO 47: Aflav_SWI5_1RGGTTTGCAATTCTTCA SEQ ID NO 48: Anig_SWI5_1 CCTGTGTGTAGCGCAGCSEQ ID NO 49: Anig_SWI5_1F ACAGTACCGCAGC SEQ ID NO 50: Anig_SWI5_1RCTTCCGGGGTGAA SEQ ID NO 51: Aterr_SWI5_1 CGATCTCTACATGTTCAACGACSEQ ID NO 52: Aterr_SWI5_1F CGAAAGCTCCCT SEQ ID NO 53: Aterr_SWI5_1RCCGTCTGCGGTC SEQ ID NO 54: Afum_SWI5_2 ATTCCTTGCTGGAGGAGAACASEQ ID NO 55: Afum_SWI5_2F GCACGATGGGACCGT SEQ ID NO 56: Afum_SWI5_2RGATTGCGAGAATTGGG SEQ ID NO 57: Afum_SWI5_3R CCGATTGCGAGAATTGGGSEQ ID NO 58: Afum_SWI5_3F ACGATGGGACCGT SEQ ID NO 59: Aflav_SWI5_2FGTCTCTACACATGCAACGATCGC SEQ ID NO 60: Aflav_SWI5_2RTGCATTACCAGGGGACCTGTT SEQ ID NO 61: Aflav_SWI5_2TGTGCAAGATGGTAACCTTCTAAAT SEQ ID NO 62: Aterr_SWI5_2FGACCATGCACGATGGTAACGTT SEQ ID NO 63: Aterr_SWI5_2R GTGGGTCCGAAACGTTGCASEQ ID NO 64: Aterr_SWI5_2 AGACGGTGATGGGAGAAGTSEQ ID NO 65: Anig_SWI5_2F ATCCATGCAAGATGGTAC SEQ ID NO 66: Anig_SWI5_2RCTACACACAGGTATCGTT SEQ ID NO 67: Anig_SWI5_2 TGCTCGGGGACAGCCAA. fumigatus sequence information SEQ ID NO 68: >Fum505TTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCATTGCTATGGGAAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACAGCSEQ ID NO 69>Fum359TTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCTTTGCTATGGGAAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACAGCSEQ ID NO 70>Fum2010TTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCTTTGCTATGGGAAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACAGCSEQ ID NO 71>FUM4185TTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCTTTGCTATGGGAAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACASEQ ID NO 72>Fum419TTTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGATGGGACCGTTTCCATTACTAACGCAACAGCAACTCAGCAGCACCAAATCCTTGCTGGAGGAGAACAGCATCCCCAATTCTCGCAATCGGCACATTTCCCCCAGCACTCCTCTCCCATGCCCGTGATGCCTGAATGCCCGTCTTTGACCTCAGAAGACTTGCAAGCATTATCCAATTCTACCAGCAATGCGAATCATCCGGGCATGGCTTACATGAATTCGAGCTTCATTGCTATGGGAAATCCGAGCTTGGGACTTCGACCAATGGATAACAATCTGAATCTGATGCAACAGCAACAGCA. flavus sequence information SEQ ID NO 73>flav2008TTTCAACACCTTCCGCTCTTGATGCCGCCAAAGTCCCCAGTCTTCCTGCCCAGGCAATGCACCGATACCATGCTCACCGTCGCGGCCAGAGCCTAGACCAGAGGTCTCTACACATGCAACGATCGCAGACCGTGCAAGATGGTAACCTTCTAAATACTAACGCAACAGGTCCCCTGGTAATGCAACAGCAACACTATGCTCGTTCGGCGCAACCGACACCCATGCCCATGATGCCTGAGTGCCAGACTTTCAGTCCTGAAGAATTGCAAACCCAACCAAGTATGGGATACATGAGCCCAGCCTTCGCCAAGGCCGAGACCCCGGCGCTGGAGAGTCGGCCGATGAACCTCCATCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 74>FLAv117.62TTTCAACACCTTCCGCTCTTGATGCCGCCAAAGTCCCCAGTCTTCCTGCCCAGGCAATGCACCGATACCATGCTCACCGTCGCGGCCAGAGCCTAGACCAGAGGTCTCTACACATGCAACGATCGCAGACCGTGCAAGATGGTAACCTTCTAAATACTAACGCAACAGGTCCCCTGGTAATGCAACAGCAACACTATGCTCGTTCGGCGCAACCGACACCCATGCCCATGATGCCTGAGTGCCAGACTTTCAGTCCTGAAGAATTGCAAACCCAACCAAGTATGGGATACATGAGCCCAGCCTTCGCCAAGGCCGAGACCCCGGCGCTGGAGAGTCGGCCGATGAACCTCCATCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 75>FLAV110.55TTTCAACACCTTCCGCTCTTGATGCCGCCAAAGTCCCCAGTCTTCCTGCCCAGGCAATGCACCGATACCATGCTCACCGTCGCGGCCAGAGCCTAGACCAGAGGTCTCTACACATGCAACGATCGCAGACCGTGCAAGATGGTAACCTTCTAAATACTAACGCAACAGGTCCCCTGGTAATGCAACAGCAACACTATGCTCGTTCGGCGCAACCGACACCCATGCCCATGATGCCTGAGTGCCAGACTTTCAGTCCTGAAGAATTGCAAACCCAACCAAGTATGGGATACATGAGCCCAGCCTTCGCCAAGGCCGAGACCCCGGCGCTGGAGAGTCGGCCGATGAACCTCCATCTCAATCTGATGCAACAGCAACAGC A. niger sequence informationSEQ ID NO 76>Nig2864TCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGTTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAATCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCGTGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGAACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCGACCTGATGCAACAACAACAG ASEQ ID NO 77>Nig554TCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGCTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAATCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCATGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGGACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCGATCTGATGCAACAACAACAG ASEQ ID NO 78>Nig2828TCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGTTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAACCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCGTGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGGACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCGACCTGATGCAACAACAACAG ASEQ ID NO 79>Nig2599TCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGTTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAATCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCGTGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGGACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCGACCTGATGCAACAACAACAG ASEQ ID NO 80>Nig121TCGACGCCGTCCGCTCTTGATGCCGTGAAAGCCCCCAGCCTTCCGGCGCAAGCGATGCATCGTTATCATGCCCATCGTCGAGGACAGAGTTTTGACAACAGAGCTTTGCGCGTCCAGCGATCGCAATCCATGCAAGATGGTACAAATCATACTACTAACTCTACAGTACCGCAGCAGCACCATTCGAATATGCTCGGGGACAGCCAACACCAACGATACCTGTGTGTAGCGCAGCCGTCGTTTCCCCAGCAATCAGCCCCCATGCCCGTGATCCCCGACTGTTTCACCCCGGAAGAGGTGCAAAACCTTCAAAGTCACAATGGCCAGGACAGTCAACCAAGCATGGCCTACCTGAATGCGCCCTTCGCAAAGGACGTTCCGCACATGAACATGCAGTTCGACCTGATGCAACAACAACAG AA. terreus sequence information SEQ ID NO 81>Terr583TTTCCACCCCTTCTGCCATTGACGCCGCGAAAGCTCCCTCCCTGCCGGCGCAGGCATTGCATCGATACCATGCTCATCGCCGAGGCCAGAGTTTCGACCAACGATCTCTACATGTTCAACGACCGCAGACCATGCACGATGGTAACGTTTCAGCTACTAACAACACAGGACCGCAGACGGTGATGGGAGAAGTGCAGCAGCTGCAACGTTTCGGACCCACAGGACACTCAGGCTATCACCAACACTCGGCTTCTATGCCGGTAATGCCCGAGTGCCAACCATTGAGCCAGGAAGACTTCCAGACCCTGGGCAATCGCAATGTCCCAGACAACCAGACCGCCATGACCTATATGACGCCCGCGCTGCACCTCAACATGATGCAG SEQ ID NO 82>Terr5677TTTCCACCCCTTCTGCCATTGACGCCGCGAAAGCTCCCTCCCTGCCGGCGCAGGCATTGCATAGATACCATGCTCATCGCCGAGGCCAGAGTTTCGACCAACGATCTCTACATGTTCAACGACCGCAGACCATGCACGATGGTAACGTTTCAGCTACTAACAACTCAGGACCGCAGACGGTGATGGGAGAAGTGCAGCAGCTGCAACGTTTCGGACCCACAGGACACTCAGGCTATCACCAACACTCGGCTTCTATGCCGGTAATGCCCGAGTGCCAACCATTGAGCCAGGAAGACTTCCAGACCCTGGGCAATCGCAATGTCCCAGACAACCAGACCGCCATGACCTATATGACGCCCGCGCTGCACCTCAACATGATGCAG SEQ ID NO 83>Terr307TTTCCACCCCTTCTGCCATTGACGCCGCGAAAGCTCCCTCCCTGCCGGCGCAGGCATTGCATCGATACCATGCTCATCGCCGAGGCCAGAGTTTCGACCAACGATCTCTACATGTTCAACGACCGCAGACCATGCACGATGGTAACGTTTCAGCTACTAACAACACAGGACCGCAGACGGTGATGGGAGAAGTGCAGCAGCTGCAACGTTTCGGACCCACAGGACACTCAGGCTATCACCAACACTCGGCTTCTATGCCGGTAATGCCCGAGTGCCAACCATTGAGCCAGGAAGACTTCCAGACCCTGGGCAATCGCAATGTCCCAGACAACCAGACCGCCATGACCTATATGACGCCCGCGCTGCACCTCAACATGATGCAG N. fischeri sequence informationSEQ ID NO 84>FISCH19912TCTCCACCCCCAATGCGCTGGAGGCCGCCAAAGTTCCTACCCTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGGCAGAGTCTTGACCAACGTCCTCGACCTATGCACGATGGGGCCGTTTCCATTACTAACGCAACAGCAACTCAGCAGTCCCGAATCCTTGCCGGGGGAGCGCATCATCCCCGATTCTCGCAATCGGCGCATTACCCTCACCATTCCTCTCCCATGCCTGTGATGCCTGAATGCCCGTCTCTGACTTCGGAAGACTTGGAAGCATTATCCAATTCTACCAGCAACGCGACCCATCCAGGCATGGCTTACATGAATTCGAGCTTCGTTACCATGGGAAACCCGAGCTTGGGGAATCGACCAATGGATAACAATCTCAATCTGATGCAACAGCAACAGCSEQ ID NO 85>2-1085 N. fischeriTCTCCACTCCCAATGCGCTGGAGGCCGCCAAAGTTCCTACACTTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGGCAGAGTCTCGACCAACGTCCTCGACCTATGCACGATGGGGCCGTTTCCATTACTAACGCAACAGCAACTCAGCAGTCCCGAATCCTTGCCGGGGGAGCGCATCATCCCCGATTCTCGCAATCGGCGCATTTCCCTCAGCATTCCTCTCCCATGCCTGTGATGCCTGAATGCCCGTCTCTGACCTCGGAAGACTTGGAAGCACTATCCAATTCTACCAGCAACGCGAACGATCCAGGCATGGCTTACATGAATTCGAGCTTCATTCCCATGGGAAACCCGAGCTTGGGGAATCGACCAATGGACAGCAATCTCAATCTGATGCAACAGCAACAGCSEQ ID NO 86>FISCH214525TCTCAACCCCCAATGCGCTGGAAGCCGCCAAAGTTCCTACCCCTCCAGCACAGGCATTGCAGCGAATCAATGCGCATCGCCGTGGACAGAGTCTCGACCAACGACCTTTGCACGTCCGACGTCCTCGACCTATGCACGATGGGGCCGTTTCCATTACTAACGCAACAGCAACTCAGCAAACCCAAATCCTTGCCGGGGGAGCGCAACATCCCCGATTCTCGCAATCGGCGCATTTTCCCCAGCATTCCTCTCCCATGCCTGTAATGCCTGAGTGCCCATCTTTGACCTCGGAAGACTTGCAAGCATTGTCCAATTCTACCAGCAATGCGAACCATCCGGGCATGGCTTACATGAATTCGAGCTTCATTACCATGGGAAACCCGAGCTTGGGGATCCGACCAATGGACAACAATCTCAATCTGATGCAACAGCAACAGC A. clavatus sequence informationSEQ ID NO 87>CLAV5138TCTCAACCCCCAATGTCCTGGAAGCCGTCAAAGTACCTACTCTCCCGGCGCAGGCTTTGCAGCGCATCCAGGCGCATCGTCGGGGACAGAGTCTCGATCAGCGATCTGTGCATGCCCAACGATCTCGTCCCATGCAAGATGGTGGTCCTTCCATTACTAACCCAGCAGTGCCTCAGCAACCCCAGATGGTTGCCGGGGGAGCGCCTCATCAGCAATTCCCTCAATCGTCGCAATTCCCCCAGCAACTTACCCCCATGCCTATGATGCCCGAATGTCAGTCGTTTCCCTCCGACGAGTTGCAGGCGTTGTCCGGACAGAGCATCAACGTGAATCAACCGGACATGGCTTATATGATTCCAGACTTCGTCAACATCGGAAATCATTGCGTTGGGAACCGACCCATGGTCAGCAACCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 88>CLAV1348TCTCAACCCCCAATGTCCTGGAAGCCGTCAAAGTACCTACTCTCCCGGCGCAGGCTTTGCAGCGCATCCAGGCGCATCGTCGGGGACAGAGTCTCGATCAGCGATCTGTGCATGCCCAACGATCTCGTCCCATGCAAGATGGTGGTCCTTCCATTACTAACCCAGCAGTGCCTCAGCAACCCCAGATGGTTGCCGGGGGAGCGCCTCATCAGCAATTCCCTCAATCGTCGCAATTCCCCCAGCAACTTACCCCCATGCCTATGATGCCCGAATGTCAGTCGTTTCCCTCCGACGAGTTGCAGGCGTTGTCCGGACAGAGCATCAACGTGAATCAACCGGACATGGCTTATATGATTCCAGACTTCGTCAACATCGGAAATCATTGCGTTGGGAACCGACCCATGGTCAGCAACCTCAATCTGATGCAACAGCAACAGC SEQ ID NO 89>CLAV7944TCTCAACCCCCAATGTCCTGGAAGCCGTCAAAGTACCTACTCTCCCGGCGCAGGCTTTGCAGCGCATCCAGGCGCATCGTCGGGGACAGAGTCTCGATCAGCGATCTGTGCATGCCCAACGATCTCGTCCCATGCAAGATGGTGGTCCTTCCATTACTAACCCAGCAGTGCCTCAGCAACCCCAGATGGTTGCCGGGGGAGCGCCTCATCAGCAATTCCCTCAATCGTCGCAATTCCCCCAGCAACTTACCCCCATGCCTATGATGCCCGAATGTCAGTCGTTTCCCTCCGACGAGTTGCAGGCGTTGTCCGGACAGAGCATCAACGTGAATCAACCGGACATGGCTTATATGATTCCAGACTTCGTCAACATCGGAAATCATTGCGTTGGGAACCGACCCATGGTCAGCAACCTCAATCTGATGCAACAGCAACAGC A. nidulans sequence information SEQ ID NO 90>Nid589CGACTCCCTCCGCGCTCGATGCCGTAAAACCCCCAGGCCTTTCTCCACAGGCTCTGCAGAGATATCATGCTCATCGCCGCGGCCAAAGTCTGGACCAGCGAGCTGTACAAGCTCAAGCTCAGCGACAACAGCTCGTGCAAGATGCGTCAAGTACTAACCAAACAGCACCGCAATTCGCGCCTAACTCAACCCTCGTCCCCTTAATTCCTGACTCCCAGATCTTCGGCCAAGACGACATGCAGGCTTCAAGTCACGCCAATTACCAGACGCCTCACAGCCTACCCTACTTGCACACGAATTTTGTCAAGGCCGATGATCAGGCTCGGGATGCTCGACCTGTCAATCACCATCTCAATCTCATTCAACAGCA SEQ ID NO 91>Nid7063CGACTCCCTCCGCGCTCGATGCCGCAAAACCTCCAGGCCTTTCTCCACAGGCTCTGCAGAGATACCATGCTCATCGCCGCGGCCAAAGTCTGGACCAGCGAGCTGTGCAAGCTCAAGCTCAGCGACAACAGCTCGTGCAAGATGCGTCAAGTACTAACCAAACAGCACCGCAATTCGCGCCTAACTCAACCCTCGTCCCTTTAATGCCTGACTCCCAGATCTTCGGCCAAGACGACATGCAGGCTTCAAGTCACGCCAATTACCAGACGCCTCACAGTCTACCCTACTTGCACACGAACTTTGTCAAGGCCGATGATCAGGCTCGGGATGCTCGACCTGTCAATCACCACCTCAATCTCATTCAACAGCA SEQ ID NO 92>Nid8o8CGACTCCCTCCGCGCTCGATGCCGTAAAACCCCCAGGCCTTTCTCCACAGGCTCTGCAGAGATATCATGCTCATCGCCGCGGCCAAAGTCTGGACCAGCGAGCTGTACAAGCTCAAGCTCAGCGACAACAGCTCGTGCAAGATGCGTCAAGTACTAACCAAACAGCACCGCAATTCGCGCCTAACTCAACCCTCGTCCCCTTAATTCCTGACTCCCAGATCTTCGGCCAAGACGACATGCAGGCTTCAAGTCACGCCAATTACCAGACGCCTCACAGCCTACCCTACTTGCACACGAATTTTGTCAAGGCCGATGATCAGGCTCGGGATGCTCGACCTGTCAATCACCATCTCAATCTCATTCAACAGCA SEQ ID NO 93>Nid670CGACTCCCTCCGCGCTCGATGCCGCAAAACCTCCAGGCCTTTCTCCACAGGCTCTGCAGAGATACCATGCTCATCGCCGCGGCCAAAGTCTGGACCAGCGAGCTGTGCAAGCTCAAGCTCAGCGACAACAGCTCGTGCAAGATGCGTCAAGTACTAACCAAACAGCACCCCAATTCGCGCCTAACTCAACCCTCGTCCCTTTAATGCCTGACTCCCAGATCTTCGGCCAAGACGACATGCAGGCTTCAAGTCACGCCAATTACCAGACGCCTCACAGTCTACCCTACTTGCACACGAACTTTGTCAAGGCCGATGATCAGGCTCGGGATGCTCGACCTGTCAATCACCACCTCAATCTCATTCAACAGCA A. glaucus sequence informationSEQ ID NO 94>GLAU117314TCTCGCAACCCTACAGCCCTCGAGGCCGCGAAAGTTCCCAGTCTCCCTGCACCGGCATTGCAACGGCTCAATGCTCATCGACGAGGCCAGAGCCTCGACACACGGGCCTTCCAGATGCAACAACGAGCACAGGCCATGCAGGATGGGAATCTTTCTTTTACTAACCAAGGAACAGTACACCAACAACCACAACCACACAATGTCTTGCGCGAGGCCCAACAACAGCGATTGGCTAGACAGGGACATCAGATGTATCCCGCTAATTCAACATCTGTACCCCTGATGCCCGACTGCCACGCGTTCAGCCAAGGGGACCTGCATATGCCTGCGAACCAAGACAACAATGAGAACCACCAAAGCGCGGCGTATATTGAAGCACAGCTGAATCTGAACTTCAATCTGATGCAACAGCAACAGCSEQ ID NO 95>GLAU543TCTCGCAACCCTACAGCCCTCGAGGCCGCGAAAGTTCCCAGTATCCCTGCACCGGCATTGCAACGGCTCAATGCTCATCGACGAGGCCAGAGCCTCGACACACGGGCCTTCCAGATGCAACAACGAGCACAGGCCATGCAGGATGGGAATCTTTCTTTTACTAACCAAGGAACAGTACACCAACAACCACAACCACACAATGTCTTGCGCGAGGTCCAACAACAGCGATTGGCTAGACAGGGACATCAGATGTATCCCGCTAATTCAACATCTGTACCCCTGATGCCCGACTGCCACGCGTTCAGCCAAGGGGACCTGCATATGCCTGCGAACCAAGACAACAATGAGAACCACCAAAGCGCGGCGTATATTGAAGCACAGCTGAATCTGAACTTCAATCTGATGCAACAGCAACAGC

REFERENCES

-   Aerne B L, Johnson A L, Toyn J H, Johnston L H.    -   Swi5 controls a novel wave of cyclin synthesis in late mitosis.        Mol Biol Cell. 1998 April; 9(4):945-56.-   Akamatsu Y, Dziadkowiec D, Ikeguchi M, Shinagawa H, Iwasaki H.    -   Two different Swi5-containing protein complexes are involved in        mating-type switching and recombination repair in fission yeast.        Proc Natl Acad Sci USA. 2003 Dec. 23; 100(26):15770-5. Epub 2003        Dec. 8.-   Butler G, Thiele D J.    -   ACE2, an activator of yeast metallothionein expression which is        homologous to SWI5. Mol Cell Biol. 1991 January; 11(1):476-85.-   MacCallum D M, Findon H, Kenny C C, Butler G, Haynes K, Odds F C.    -   Different consequences of ACE2 and SWI5 gene disruptions for        virulence of pathogenic and nonpathogenic yeasts. Infect Immun.        2006 September; 74(9):5244-8.-   Ellermeier C, Schmidt H, Smith G R.    -   Swi5 acts in meiotic DNA joint molecule formation in        Schizosaccharomyces pombe. Genetics. 2004 December;        168(4):1891-8. Epub 2004 Sep. 30.

The invention claimed is:
 1. A diagnostic kit for a fungal or yeastspecies comprising an deoxyoligonucleotide probe comprising SEQ ID NO:54, or a probe having a sequence at least 95% sequence identity thereto,and wherein the probe is from about 21 to about 100 nucleotides andcapable of binding to at least a portion of the Aspergillus spp. SWI5gene (“SWI5 gene”) or its corresponding mRNA and wherein the detectablemoiety comprises a radioisotope, a fluorescent moiety, chemiluminescentlabel, a nanoparticle, an enzyme or a ligand.
 2. The kit of claim 1wherein the portion of the SWI5 gene is a portion of the region of thegene from base pair position 1 to base pair position 2319 of SEQ ID NO:29 of the Aspergillus SWI5 gene.
 3. The kit of claim 1 wherein theportion of the SWI5 gene is a portion of the region of the gene frombase pair position 38 to base pair position 472 of SEQ ID NO: 29 of, orfrom base pair position 1423 to base pair position 1627 of SEQ ID NO: 29of the Aspergillus SWI5 gene.
 4. The kit of claim 1 comprising a probefor a portion of the region of the gene from base pair position 1 tobase pair position 2319 of SEQ ID NO: 29 of, from base pair position 38to base pair position 472 of SEQ ID NO: 29, and from base pair position1423 to base pair position 1627 of SEQ ID NO: 29 of the Aspergillus SWI5gene.
 5. The kit of claim 1 further comprising a probe comprising anoligonucleotide having a sequence selected from SEQ ID NO 17, 18, 42,45, 48, 51, 61, 64, or 67 or sequences having at least 90% sequenceidentity thereto and which and wherein the probe is from about 10 toabout 100 nucleotides and capable of binding to at least a portion ofthe Aspergillus spp. SWI5 gene (“SWI5 gene”) or its corresponding mRNA.6. The kit of claim 1 further comprising a primer for amplification ofat least a portion of the SWI5 gene.
 7. The kit of claim 1 comprising aforward and a reverse primer for a portion of the SWI5 gene.
 8. The kitof claim 1 further comprising at least one forward in vitroamplification primer and at least one reverse in vitro amplificationprimer, the forward amplification primer being selected from the groupconsisting of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 36, 38, 40, 43, 46,49, 52, 55, 58, 59, 62 or 65, or sequences having at least 90% sequenceidentity thereto and which can also act as a forward amplificationprimer and the reverse amplification primer being selected from thegroup consisting of SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 37, 39, 41,44, 47, 50, 53, 56, 57, 60, 63 or 66, sequences having at least 90%sequence identity thereto and which can also act as a reverseamplification primer.
 9. A diagnostic kit as claimed in claim 8, basedon direct nucleic acid detection technologies, signal amplificationnucleic acid detection technologies, and nucleic acid in vitroamplification technologies is selected from one or more of PolymeraseChain Reaction (PCR), Ligase Chain Reaction (LCR), Nucleic AcidsSequence Based Amplification (NASBA), Strand Displacement Amplification(SDA), Transcription Mediated Amplification (TMA), Branched DNAtechnology (bDNA) and Rolling Circle Amplification Technology (RCAT) orother enzymatic in vitro amplification based technologies.
 10. The kitof claim 1, further comprising at least one reverse in vitroamplification primer of SEQ ID NO 55, or sequences having at least 90%sequence identity thereto and which can also act as a forwardamplification primer and a reverse amplification primer of SEQ ID NO 56,or sequences having at least 90% sequence identity thereto and which canalso act as a reverse amplification primer.
 11. The kit of claim 1,wherein the probe consists of SEQ ID NO 54 and further comprisingprimers consisting of SEQ ID NOs 55 and 56, or sequences having at least90% sequence identity thereto.
 12. A diagnostic kit for a fungal oryeast species comprising an deoxyoligonucleotide probe comprising SEQ IDNO: 54; wherein the probe is from about 21 to about 100 nucleotides andcapable of binding to at least a portion of the Aspergillus spp. SWI5gene (“SWI5 gene”) or its corresponding mRNA and wherein the probe isattached to a detectable moiety to detect or confirm probe hybridizationto the portion of the SWI5 gene or its corresponding mRNA.
 13. Adiagnostic kit of claim 12, further comprising SEQ ID NO: 17, 48, 51,61, 64, or 67.